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J Nat Med (2009) 63:290296 DOI 10.

1007/s11418-009-0336-6

ORIGINAL PAPER

Antinociceptive, antipyretic, and anti-inammatory activities of Putranjiva roxburghii Wall. leaf extract in experimental animals
Wantana Reanmongkol Tassanee Noppapan Sanan Subhadhirasakul

Received: 22 January 2009 / Accepted: 31 March 2009 / Published online: 23 April 2009 The Japanese Society of Pharmacognosy and Springer 2009

Abstract The effects of the ether extract from the leaves of Putranjiva roxburghii (P. roxburghii) Wall. were assessed on nociceptive responses in mice by using writhing, hot plate, and formalin tests and the antipyretic activity was determined in yeast-induced fever in rats. Anti-inammatory activities were also investigated using carrageenininduced paw edema in rats and croton oil-induced ear and anus edemas. The ether extract (100, 200, and 400 mg/kg, p.o.) of P. roxburghii dose-dependently produced analgesic activity in acetic acid-induced writhing in mice. The extract had no signicant effect in the hot plate test in mice. At the dose of 400 mg/kg, the extract signicantly suppressed the licking activity in the late phase of the formalin test in mice and decreased fever induced by yeast in rats. The extract exhibited moderate inhibitory activity of inammation in carrageenin-induced paw edema in rats. The extract inhibited croton oil-induced ear edema in a dose-dependent manner (1.25, 2.5, and 5.0 mg/ear) in mice. The extract decreased anus edema induced by croton oil at the high dose of 800 mg/kg in rats. The results indicated that the ether extract of P. roxburghii leaves possesses analgesic, antipyretic, and anti-inammatory activities. Keywords Putranjiva roxburghii Pain Fever Inammation Experimental animals
W. Reanmongkol (&) T. Noppapan Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand e-mail: wantana.r@psu.ac.th S. Subhadhirasakul Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand

Introduction Putranjiva roxburghii Wall. (P. roxburghii) or Drypetes roxburghii (Wall.) Hurusawa, locally known as Ma Kham Kai or Pra Kham Kai, belongs to the family Euphorbiaceae. It is widely grown in Thailand, Nepal, Bangladesh, India, Indochina, Myanmar, and Sri Lanka [1]. In Thai folklore medicine, its leaves and fruits have been traditionally used for the treatment of fever, muscle sprain, arthralgia, and rheumatism [1, 2], and the whole plant of P. roxburghii has also been used for the treatment of fever and hemorrhoids [1]. Two triterpenoids, namely putranjivanonol and putranjic acid, were isolated from the trunk bark of P. roxburghii [3]. The isolation of four other triterpenoids, friedelin, putranjivadione, friedelanol, and roxburgholone, from the bark of P. roxburghii has also been described [4]. Roxburghonic acid, a triterpene acid, and putraavone, a biavonoid, were isolated from the alcoholic extract of P. roxburghii leaves [5, 6]. Although P. roxburghii has been used as traditional medicine, no in vivo pharmacological studies have been conducted regarding the antinociceptive, antipyretic, and anti-inammatory actions of this plant. Thus, the aim of this study was to investigate the antinociceptive, antipyretic, and anti-inammatory effects of the extract from the leaves of P. roxburghii.

Materials and methods Plant material The air-dried leaves of P. roxburghii Wall. (Euphorbiaceae) were purchased from herbal drugstores in Songkhla, Thailand. Authentication of plant material was carried out at the Department of Pharmacognosy and Pharmaceutical

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Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand, where the herbarium voucher (No. SS 2549-001) has been kept. The air-dried leaves of P. roxburghii were pulverized into powder by using a blender and stored in an airtight container. Preparation of the extract from the leaves of P. roxburghii The air-dried leaves of P. roxburghii (1.2 kg) were macerated with 95% ethanol at room temperature and then ltered and evaporated to give a syrupy mass. The marc was remacerated with ethanol four times, ltered, and evaporated. All syrup masses were combined and exhaustively extracted with ether, and the ethereal solution thus obtained was separated into acidic and neutral fractions by washing with 0.5% NaOH solution. The alkali-soluble fraction was acidied, extracted with ether, and evaporated to give a syrupy mass of ether extract (84 g or 24% yield). The ether extract was subjected to column chromatography using a gradient elution of MeOH/CHCl3 (100% CHCl3 and 2, 5, 10, 50% MeOH/CHCl3), and was monitored by TLC (avonoid-type compounds were visualized rst under UV light and then by spraying the TLC plate with an appropriate reagent and heating). The ether extract was used as the test extract. All doses were expressed in terms of crude extract (mg/kg body weight) unless otherwise specied. Animals Animals used in this study were male Swiss albino mice weighing 2535 g and male Wistar rats weighing 150 200 g. All animals were obtained from the Southern Laboratory Animal Facility, Prince of Songkla University, Hat Yai, Songkhla, Thailand. The animals were housed for at least 1 week in the laboratory animal room prior to testing. Food and water were given ad libitum unless otherwise specied. All experimental protocols were approved by the Animal Ethics Committee, Prince of Songkla University (No. 0521.11/166). Antinociceptive activities Writhing test Writhing behavior was tested as previously reported [7, 8]. Thus, 0.6% acetic acid solution (10 ml/kg) was injected intraperitoneally in mice and the number of writhings and stretchings was counted over a 20-min period. The ether extract of P. roxburghii (EPR; 100, 200, and 400 mg/kg), a reference analgesic drug aspirin (200 mg/kg), or cosolvent (control) were orally administered 30 min before acetic acid.

Hot plate test The hot plate test was carried out according to the method described by Woolfe and MacDonald [9]. Mice were placed on a hot plate maintained at 55 1C. Latency of nociceptive response such as licking of a hind limb or jumping was measured. Starting 30 min after oral administration (p.o.) of the test agents, or 15 min after subcutaneous (s.c.) injection of morphine sulfate, the nociceptive response was measured every 15 min over a 60-min period. The cutoff time was 45 s. Only the mice that showed nociceptive responses within 15 s were used for the experiment. Formalin test Thirty minutes after oral administration of EPR (100, 200, and 400 mg/kg), aspirin (200 mg/kg), or cosolvent (control), or 15 min after subcutaneous injection of morphine, 20 ll of 2.5% formalin in saline was injected subcutaneously into a hind paw of the mice. The time spent licking the injected paw was recorded and the data were expressed as total licking time in the early phase (05 min) and the late phase (1530 min) after formalin injection [10]. Antipyretic activity Yeast-induced fever Antipyretic activity of drug was measured by slightly modifying the method described by Adams et al. [11]. Male Wistar rats were fasted overnight with water ad libitum before the experiments. Pyrexia was induced by subcutaneously injecting 20% (w/v) brewers yeast suspension (10 ml/kg) into the animals dorsum region. Seventeen hours after the injection, the rectal temperature of each rat was measured using a digital thermometer (SK-1250 MC, Sato Keiryoki Mfg. Co., Ltd, Japan). Only rats that showed an increase in temperature of at least 0.7C were used for the experiments. Cosolvent vehicle, EPR (100, 200, and 400 mg/kg), or aspirin (200 mg/kg) was administered orally and the temperature was measured at 1, 2, 3, 4, and 5 h after drug administration. Anti-inammatory activities Carrageenin-induced paw edema According to the method described by Winter et al. [12], the initial right hind paw volume of the rats was measured using a plethysmometer (Ugo Basile) and then 0.1 ml of 1% (w/v) carrageenin was subcutaneously injected into the subplantar region of the right hind paw. The volume of

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right hind paw was measured at 1, 2, 3, 4, and 5 h after carrageenin injection, and the edema volume was determined. Cosolvent, EPR (100, 200, and 400 mg/kg), or aspirin (200 mg/kg) was orally administered 30 min before carrageenin injection. Croton oil-induced ear edema Edema was induced according to the method described by Tubaro et al. [13]. Cutaneous inammation was induced by application of 10 ll of an acetone solution containing the irritant (5% croton oil) to the inner surface of the right ear of the mice. The left ear received an equal volume of acetone. EPR (1.25, 2.5, and 5.0 mg/ear) was applied topically to the right ear 1 h before application of croton oil. The left ear received the vehicle. Indomethacin (1 mg/ ear) was used as a reference anti-inammatory. Four hours after the application of the irritant agent, the mice were sacriced and a plug (7-mm diameter) was removed from both the treated and untreated ears. The edematous response was measured as the weight difference between the two plugs. Croton oil-induced anus edema Croton oil-induced anus edema was measured by modifying the method described by Nishiki et al. [14]. A cotton swab soaked with the inducer, which consisted of 6% croton oil in diethyl ether (0.2 ml), was inserted into the anus of rats for 10 s. One hour after administration of croton oil, cosolvent or EPR (100, 200, 400, and 800 mg/ kg), or aspirin (200 mg/kg) was orally administered once daily for 3 days. On the fourth day, rats were anaesthetized, then the size of each rats anus (mm) was measured by using a vernier caliper. Chemicals The following drugs were used: morphine sulfate, brewers yeast, carrageenin lambda and croton oil (Sigma Chem. Co., St. Louis, USA), aspirin and Tween 80 (Srichand United Dispensary Co., Ltd, Bangkok, Thailand), indomethacin (Fluka BioChemika, Japan), sodium chloride (Carlo Erba, Germanny), acetic acid (J.T., Baker Inc., Phillipsburg, USA), propylene glycol and acetone (Vidhyasom Co., Ltd., Bangkok, Thailand), ethanol (Merck KGaA, Germany), and ether (Labscan Asia Co., Ltd, Bangkok, Thailand). The extract was dissolved in cosolvent (containing propylene glycol, Tween 80 and water) and administered orally in a constant volume (10 ml/kg for mice and 5 ml/kg for rats) 30 min before the experiments unless otherwise specied. Morphine sulfate was dissolved in 0.9% sodium chloride solution and administered

subcutaneously. All drug solutions were prepared immediately before starting the experiments. Statistical analysis Data were expressed as mean SEM and were analyzed statistically by one-way ANOVA procedures, followed by Bonferronis test. A difference was considered signicant at P \ 0.05.

Results Effects of EPR on nociceptive responses in mice Writhing test Oral administration (100, 200, 400 mg/kg) of EPR dosedependently reduced the number of writhings and stretchings induced by intraperitoneal 0.6% acetic acid in mice. At the dose of 400 mg/kg, EPR inhibited the writhing by 61%. The reference drug aspirin (200 mg/kg) also produced signicant protective effects towards the acetic acidinduced pain: 69% inhibition, compared with the control group (Table 1). Hot plate test Neither EPR (100, 200, and 400 mg/kg, p.o.) nor aspirin (200 mg/kg, p.o.) signicantly exerted protective effects on heat-induced pain in mice. In contrast, a centrally acting analgesic drug, morphine sulfate (10 mg/kg, s.c.), markedly increased pain latency in the hot plate test, compared with the control group (data not shown). Formalin test EPR dose-dependently decreased the licking activity in the late phase but not in the early phase of formalininduced pain. At the dose of 400 mg/kg, EPR signicantly inhibited the licking activity in the late phase by 58% (Table 2). The reference drug aspirin (200 mg/kg) also suppressed pain only in the late phase at the same inhibition level (58%). In contrast, the reference drug morphine sulfate (10 mg/kg) markedly reduced the licking activity in both phases of formalin-induced nociception in mice by 58 and 95% inhibition, respectively, compared with the control group. Effects of EPR on yeast-induced fever in rats EPR signicantly suppressed the pyrexia induced by yeast in rats at the dose of 400 mg/kg at 1, 2, 3, 4, and 5 h after

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oral administration. Aspirin (200 mg/kg) also signicantly decreased the fever induced by yeast in rats, compared with the control group (Table 3). Effects of EPR on inammation Carrageenin-induced paw edema in rats EPR at the dose of 400 mg/kg signicantly suppressed the carrageenin-induced paw edema at 3, 4, and 5 h after carrageenin injection. Aspirin (200 mg/kg) reduced

carrageenin-induced paw edema in rats at 1, 2, 3, 4, and 5 h, compared with the control group (Table 4). Croton oil-induced ear edema in mice Topically applied EPR (1.25, 2.5, and 5.0 mg/ear) dosedependently suppressed the ear edema induced by croton oil in mice. At the dose of 5.0 mg/ear, EPR reduced the ear swelling by 42%. Indomethacin (1.0 mg/ear), the standard reference, also signicantly reduced ear edema in this test by 57%, compared with the control group (Table 5). Croton oil-induced anus edema in rats

Table 1 Effect of the ether extract from P. roxburghii leaves and aspirin on acetic acid-induced writhing response in mice Drug Control Aspirin P. roxburghii Dose (mg/kg, p.o.) 200 100 200 400 Number of writhings (counts/20 min) 38.1 1.3 11.8 1.4* 22.1 1.5* 18.5 1.4* 15.0 2.4* Inhibition (%) 0 69.0 42.0 51.5 60.6

EPR signicantly decreased the size of rat anus on croton oil-induced anus edema after oral administration but only at the high dose of 800 mg/kg. Aspirin (200 mg/kg), the reference drug, also signicantly reduced the size of anus in rats, compared with the control group (Table 6).

Discussion The results demonstrate that the ether extract obtained from the leaves of P. roxburghii (EPR) exhibited analgesic,

Each datum represents the mean SEM from ten mice *P \ 0.01 compared with the control group (Bonferronis test)

Table 2 Effect of the ether extract from P. roxburghii leaves, aspirin, and morphine on licking activity in mice Drug Dose (mg/kg, p.o.) Licking time (s) Early phase (05 min) Control Aspirin Morphine P. roxburghii 200 10 100 200 400 78.9 4.1 74.7 5.7 33.6 2.7* 82.2 9.1 86.4 6.0 73.3 5.6 Inhibition (%) 0 5.4 57.5 -4.2 -9.6 7.1 Late phase (1530 min) 74.8 11.9 31.3 4.6* 3.6 1.2* 59.8 9.3 55.4 6.5 31.7 4.5* Inhibition (%) 0 58.2 95.3 20 25.9 57.7

Each datum represents the mean of licking time SEM from ten mice in the early phase (05 min) and the late phase (1530 min) after formalin injection *P \ 0.01 compared with the control group (Bonferronis test) Table 3 Effect of the ether extract from P. roxburghii leaves and aspirin on brewers yeast-induced fever in rats Drug Dose (mg/kg, p.o.) Average rectal temperature (C) 0h Control Aspirin P. roxburghii 200 100 200 400 37.2 0.2 37.3 0.3 37.8 0.1 37.5 0.2 37.1 0.2 1h 37.1 0.1 35.7 0.2** 36.9 0.1 36.4 0.1 36.2 0.1* 2h 36.5 0.1 35.4 0.1** 36.7 0.1 36.4 0.2 35.6 0.7** 3h 36.4 0.1 35.3 0.2** 36.4 0.1 36.0 0.2 35.6 0.1* 4h 36.5 0.1 35.1 0.1** 35.9 0.8 35.8 0.2 35.3 0.2** 5h 36.8 0.2 35.0 0.5** 35.9 0.4 35.9 0.2 35.2 0.2**

Each datum represents the mean rectal temperature (C) SEM (n = 6) *P \ 0.05, **P \ 0.01 compared with the control group (Bonferronis test)

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Table 4 Effect of the ether extract from P. roxburghii leaves and aspirin on carrageenin-induced paw edema in rats Drug Dose (mg/kg, p.o.) Paw volume (ml) 0h Control Aspirin P. roxburghii 200 100 200 400 5.8 0.3 5.2 0.2 6.1 0.2 6.1 0.4 5.6 0.4 1h 7.0 0.3 5.8 0.3* 6.6 0.2 6.7 0.1 6.1 0.1 2h 7.5 0.2 6.0 0.2** 6.9 0.2 7.1 0.2 6.4 0.1 3h 8.9 0.4 6.7 0.2** 7.6 0.2 7.2 0.3 6.9 0.2* 4h 8.8 0.1 6.8 0.5** 8.0 0.4 7.9 0.4 7.0 0.2** 5h 8.6 0.2 6.4 0.2** 7.9 0.3 7.4 0.3 6.8 0.1**

Each point represents the mean SEM of six rats *P \ 0.05, **P \ 0.01 compared with the control group (Bonferronis test)

Table 5 Effect of the ether extract of P. roxburghii and indomethacin on croton oil-induced ear edema in mice Drug Control Indomethacin P. roxburghii Dose (mg/ear) 1.00 1.25 2.50 5.00 Each value represents the mean SEM from ten mice *P \ 0.01 compared with the control group (Bonferronis test) Ear swelling (mg) 13.84 0.44 6.07 0.38* 12.68 0.94 11.26 0.95 8.08 0.54* Swelling reduction (%) 57 9 19 42

Table 6 Effect of the ether extract from P. roxburghii leaves and aspirin on croton oil-induced anus edema in rats Drug Control Aspirin P. roxburghii Dose (mg/kg, p.o.) 200 100 200 400 800 Size of anus (mm) 46.5 1.0 39.5 0.7* 45.2 1.5 44.7 2.3 43.2 1.3 36.5 0.9*

Each datum represents the mean SEM from six rats *P \ 0.01 compared with the control group (Bonferronis test)

antipyretic, and anti-inammatory activities in experimental animal models. No acute toxicity was observed after oral administration of EPR even at the high dose (2 g/ kg) in mice (unpublished data). The writhing test is generally used for screening of antinociceptive activity [7, 8]. In this test, acetic acid is used as an inducer for writhing syndrome, causing pain by releasing endogenous substances including histamine, serotonin, bradykinin, substance P, and prostaglandins, which then stimulate the pain nerve endings leading to the abdominal writhing [15]. EPR showed signicant inhibition on acetic acid-induced writhing response. The reference drug aspirin (200 mg/kg) also produced signicant

protective effects towards the acetic acid-induced pain in mice. In comparison, the ether extract was slightly less potent than aspirin on nociceptive response in mice. These results indicated that the ether extract may possess antinociceptive activity. Pain induced by thermal stimuli is known to be selective to centrally but not peripherally acting analgesic drugs [16]. In the present study, morphine, a centrally acting analgesic drug, produced an inhibitory effect on the nociceptive response in the hot plate test, whereas EPR and aspirin failed to affect the responses. Therefore, this suggested that the apparently antinociceptive activity of the extract may be mediated through the peripheral mechanism(s). The formalin test is another pain model, which assesses the way an animal responds to moderate, continuous pain generated by injured tissue [17]. Centrally acting drugs such as morphine inhibited both the early and late phases, whereas peripherally acting drugs such as aspirin only inhibited the second phase [1820]. EPR also produced signicant reduction of licking activity in the late phase at the dose of 400 mg/kg but did not affect the responses in the early phase. These results also supported the hypothesis that the antinociceptive action of EPR may be mediated by a peripheral mechanism. EPR signicantly reduced the pyrexia induced by yeast in rats. The reference drug aspirin also suppressed the yeast-induced fever in rats by inhibiting the synthesis of

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prostaglandin E2 [21, 22]. These results support the use P. roxburghii leaves as an antipyretic for the treatment of fever in Thai folk medicine preparations. Carrageenin-induced paw edema, an acute inammatory model, involves several mediators released in sequence. An initial phase during the rst 1.5 h, is caused by the release of histamine and serotonin; a second phase is mediated by bradykinin from 1.5 to 2.5 h; and the third phase, in which the mediator is suspected to be prostaglandin, occurs from 2.5 to 6 h after carrageenin injection [23]. In the present study, EPR at the dose of 400 mg/kg signicantly suppressed the paw edema induced by carrageenin in rats at the third phase, suggesting the possible mechanism of action of P. roxburghii extract may involve inhibition of prostaglandin biosynthesis. Croton oil-induced ear edema is a useful model for testing topical anti-inammatory activity [24]. Topically applied EPR (5.0 mg/ear), as well as a reference drug indomethacin, signicantly reduced the inammatory response caused by croton oil. Since 12-O-tetradecanoylphorbol-13-acetate (TPA), a kind of phorbol ester present in croton oil, has been reported to stimulate phospholipid-dependent protein kinase C [25, 26] and this enzyme appears to be involved to the activation of cellular functions via protein phosphorylation. Of particular note is that the ether extract showed effective anti-inammatory activity when applied on the skin, which could be ratioalized by cutaneous absorption of the extract. In croton oil-induced anus edema, this model involves edema, inltration of inammatory cells, vasodilation, and destruction of the mucosal epithelium [14]. EPR decreased anus swelling but needed a higher dose (800 mg/kg) than those of other systemic models, as this model may produce very large amounts of inammatory tissue. The extract of P. roxburghii may be benecial for the treatment of hemorrhoids which are partly involved in anus inammation. Flavonoids or bioavonoids are known to possess antiinammatory properties [27], and avonoid compounds have been isolated from the P. roxburghii leaves [5, 6]. In the present study, our qualitative analysis also showed the presence of avonoids in EPR, which may therefore be responsible for the activities of the P. roxburghii extract. Based on these results, we concluded that EPR possesses analgesic, antipyretic, and anti-inammatory activities in acute as well as topical models which support the traditional uses of P. roxburghii for the treatment of muscle pain, fever, and inammation in Thai folk medicine preparations. However, further investigations are needed to clarify the mechanism of action of the P. roxburghii extract.
Acknowledgments The authors are very grateful to the Graduate School, Prince of Songkla University for nancial support of this work.

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