J Acupunct Meridian Stud 2009;2(4):280−287

RESEARCH ARTICLE

Anti-inflammatory and Analgesic Effects of Ethanolic Extract

Mun Fei Yam1,2*, Lee Fung Ang1, Omar Ziad Ameer1, Ibrahim Muhammad Salman1, Hesham Abdul Aziz1, Mohd. Zaini Asmawi1

1School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, Pulau Pinang, Malaysia 2Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor Darul Ehsan, Malaysia

Received: Apr 29, 2009 Abstract Accepted: Sep 29, 2009 Elephantopus tomentosus is widely used in , especially in Malaysia, for the treatment of pain and inflammation. In the present study, the analgesic and anti- KEY WORDS: inflammatory effects of a 95% ethanol extract of E. tomentosus were investigated analgesic effect; in different experimental models. In the anti-inflammation study, 1000 mg/kg of < anti-inflammatory; extract significantly reduced carrageenan-induced hind paw edema (p 0.05) and inhibited abdominal permeability compared with control (p < 0.01). The analgesic Elephantopus tomentosus activity was assayed in several experimental models in mice: (1) hot plate, (2) tail flick, (3) writhing test; and rats: carrageenan-induced hyperalgesia pain threshold test. However, at the doses tested, no significant activity was found in the hot plate test and the tail flick test. E. tomentosus ethanol extract at 1000 mg/kg sig- nificantly (p < 0.05) increased hyperalgesia pain threshold and inhibited writhing activity. The results suggest that E. tomentosus ethanol extract at 1000 mg/kg dose is effective in anti-inflammatory and non-steroidal anti-inflammatory drug type anti-nociception activities.

1. Introduction therapeutic treatment of those pathologies associ- ated with inflammatory reaction. However, the side Inflammation is the most common biological re- effects of the currently available anti-inflammatory action to a variety of stimuli and local injury [1]. drugs pose a major problem in their clinical use. Inflammatory reactions can be triggered by phys- For instance, some non-steroidal anti-inflammatory ical or chemical trauma, invading organisms and drugs (NSAIDs) may cause gastric ulceration and renal antigen-antibody reactions, and is often exacer- damage [3,4]. Owing to safety concerns associated bated by the resultant swelling or edema of tissue, with the use of synthetic anti-inflammatory and an- pain (due to increased pressure in tissues during algesic agent, the public prefer to take natural anti- edema formation or by inflammatory mediators) inflammatory and analgesic treatments from edible or even cell damage [2]. Thus the employment materials such as fruits, spices, herbs and vegetables. of anti-inflammatory agents may be helpful in the Therefore, the development and utilization of more

*Corresponding author. Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia. E-mail: [email protected]

©2009 Korean Pharmacopuncture Institute Anti-inflammatory and analgesic effects of E. tomentosus 281 effective anti-inflammatory and analgesic agents 2.3. Animals of natural origin are desired. Members of the Elephantopus genus are widely Male and female Sprague Dawley (SD) rats (age, used in Southeast Asian folk medicine. Ele- 8−12 weeks; weight, 150−200 g) and Institute phantopus spp is believed to have anti-pyretic, Charles River strain (ICR) mice (age, 3−4 weeks; anti-inflammatory and emollient activities [5]. In weight, 25−32 g) were used for the experiments. Malaysia, E. tomentosus (Et) is taken internally as The animals were kept under stan dard condi- a diuretic, febrifuge, analgesic, anti-helminthic and tions (26−30ºC) with free access to food (normal anti-inflammatory agent. Et is also applied exter- laboratory chow, Gold Coin Feed Mills Sdn Bhd, nally as a poultice for abdominal pains. However, Malaysia) and tap water. The animals were fasted very few scientific studies on Et have been con- for 6 hours prior to the experiments and acclima- ducted. Recently, Et was found to exhibit antio- tized to laboratory conditions for 7 days before xidant properties and a liver protective effect on commencement of experiments. All the experi- carbon tetrachloride-induced liver toxicity [6]. With mental procedures on animals were carried out this background, the present study aimed to investi- in accordance with the regulations and guidelines gate the analgesic and anti-inflammatory effects of of the Animal Ethics Committee, Universiti Sains E.tomentosus ethanol extract (EtE) using different Malaysia. pharmacological experimental models in an attempt to validate its ethnomedical use. 2.4. Acute toxicity

Organisation for Economic Co-operation and De- 2. Materials and Methods velopment guidelines for testing of chemicals were adhered. Healthy female SD rats were subjected 2.1. Materials to tests according to the acute oral toxicity up- and-down procedure (limit test). All experimental Carrageenan type IV (Carrageenan), sodium hydrox- animals were maintained under close observation ide (NaOH), ferric chloride (FeCl3), potassium chlo- for 14 days. ride, sodium chloride (NaCl), potassium hydroxide (KOH), Chicago sky blue, and indomethacin were 2.5. Carrageenan-induced rat hind purchased from Sigma-Aldrich (St. Louis, MO, USA). paw edema Carboxymethylcellulose (CMC) was supplied from British Drug House (Poole, England). Ethanol was The anti-inflammatory activity was evaluated ac- purchased from Riedel-de Haën (Seelze, Germany). cording to the method described by Winter et al Mayer reagent was obtained from Fluka Chemie GmbH (1962) [7]. A total of 30 male rats were used in the (Buchs, Switzerland). Glacial acetic acid, hydro- experiment. They were divided into five groups of chloric acid (HCl) and sulfuric acid (H2SO4) were six rats each. Carrageenan was freshly prepared as obtained from Merck (Darmstadt, Germany). Mor- 1% (w/v) suspension in sterile 0.9% NaCl before the phine sulfate was purchased from Remedi Pharma- experiment. A volume of 0.1 mL of carrageenan was ceutical (Selangor, Malaysia). injected into the plantar tissue of the rat right hind paw. EtE (250, 500, and 1000 mg/kg) and indometh- 2.2. Preparation of extract acin (10 mg/kg) were administered by oral gavage 1 hour before carrageenan injection. The animals in The plant was obtained from Permatang Damar the control group received 1% of CMC in distilled Laut, Penang, Malaysia. The plant was sent to the water. The footpad thickness was measured simply Herbarium, School of Biological Sciences, Universiti by placing the unanesthetized animal foot between Sains Malaysia for identification. The herbarium the anvil and spindle of peacock dial thickness voucher number is 10832. After identification, the gauge of a micrometer (Ozaki Ltd., Japan) before plant was dried in an oven (Memmert, Germany) at injection and 1, 3 and 5 hours after the injection of 45ºC and ground into powder. The powered plant carrageenan. The percentage in thickness changes material (250 g) was put into a thimble and extracted of the hind paw was calculated according to the with 5 L of 95% ethanol in a soxhlet extractor for following formula: 3 days. The extract was concentrated using Büchi RE121 rotary evaporator (Büchi Labortechnik AG, % increase in thickness = (Ct − C0)/C0 ˜ 100 Switzerland) and subsequently dried in a Hetovac VR-1 freeze dryer (Heto Lab. Equipment AS, Ct is the thickness of hind paw at t hour. C0 is the Denmark). The yield (EtE) obtained was found to thickness of hind paw before carrageenan was be 14% (w/w) of the dried plant. injected. 282 M.F. Yam et al

2.6. Acetic acid-induced peritoneal taken 1 hour after administration of EtE (250, 500, capillary dye leakage test and 1000 mg/kg, p.o.), morphine sulfate (8 mg/kg, s.c.) and vehicle (10 mL/kg of 1% CMC, p.o.). For The capillary permeability or dye leakage test was the purpose of calculation, an animal not respond- performed by modifying a previously reported ing by the cut-off time of 10 seconds was arbitrar- method (Whittle, 1964) [8]. The ICR mice (four ily assigned a response time of 10 seconds. males and four females in each group) were given EtE (250, 500 and 1000 mg/kg) and indomethacin 2.7.3. Carrageenan-induced rat hind paw (10 mg/kg). The control group was orally given 1% hyperalgesia pain threshold test CMC in distilled water. After 25 minutes, each ani- method mal was given an intravenous injection of 0.1 mL of 4% solution of Chicago sky blue in normal saline. The hyperalgesia pain threshold of the inflamed rat’s After 30 minutes, 0.2 mL of 0.25% (v/v) glacial ace- right hind paw was investigated by subjecting the tic acid solution in normal saline was injected in- hind paw to a constant force following the method traperitoneally. After an additional 20 minutes, of Winter and Flataker [9]. The various treatments the mice were killed by cervical dislocation, and [EtE (250, 500, and 1000 mg/kg, p.o.), indomethacin the visceral organs were subsequently exposed. A (10 mg/kg, p.o.) and 1% CMC (10 mL/kg, p.o.)] were period of 1 minute was allowed for blood to drain respectively given 2 hours after a sub-plantar injec- away from the abdominal wall and jugular vein. tion of 0.1 mL of 1% carrageenan. Pain threshold was The animals were then held by a flap of the ab- measured mechanically using a Letica Analgesy-meter dominal wall and the exposed visceral organs were LI7306 (Letica Scientific Instruments, Spain) before continuously irrigated with distilled water in a the injection of carrageenan and then at 4 and 6 hours petri dish. The combined washings were filtered post-injection. The pain threshold (g) was measured through glass wool and filled a 10 mL volumetric when the rat withdrew its hind paw, struggled or vo- flask. Then, 0.1 mL of 0.1 N NaOH was subsequently calized [10]. The percentage change of pain threshold added to deproteinize the filtrate. The absorbance was calculated according to the following formula: was measured using a Hitachi U-2000 spectropho- tometer at 590 nm (Hitachi, Japan). The total con- % change of pain = (Pt − P0)/P0 ˜ 100 centration of Chicago sky blue was determined as μg/10 mL from a standard calibration curve. Pt is the pain threshold at t hour and P0 is the pain threshold before carrageenan was injected. 2.7. Analgesic studies 2.7.4. Acetic acid-induced writhing test 2.7.1. Hot plate test The method of Koster et al [11] was used in this The mice were placed on an Ugo Basile 7280 hot study. EtE (250, 500 and 1000 mg/kg), paracetamol plate (Ugo Basile, Italy), which was maintained at (100 mg/kg) and 1% CMC were orally administered 55 ± 0.5ºC. The time between placement and licking to respective groups of mice. One hour later, each of the paws or jumping was recorded as latency sec- mouse was injected with 0.6% (v/v) acetic acid ond. The latency was recorded at 15, 30, 45, 60 (10 mL/kg, i.p.). The number of writhings during and 90 minutes after administration of EtE (250, 500, the 15 minutes was recorded. and 1000 mg/kg, p.o.), morphine sulfate (8 mg/kg, s.c.) and vehicle (10 mL/kg of 1% CMC, p.o.) [2]. 2.8. Phytochemical analysis Morphine sulfate was used as a reference standard. Preliminary phytochemical analysis of the extract 2.7.2. Tail flick test was performed using the following method: Alkaloids: 1 mL of 1% HCl (v/v) was added to 3 mL of The nociception was assessed using an Ugo Basile plant extract in a test tube. The mixture was heated 7360 tail flick apparatus (Ugo Basile, Italy). Briefly, for 20 minutes, cooled and filtered. Two drops of the tail flick test response was done by measuring Mayer’s reagent was added to 1 mL of the filtrate. the time taken to withdraw the tail from the heat A creamy precipitate indicates the presence of source [2]. Each mouse was encased in a small alu- alkaloids in the extract [12]. minum chamber with the middle portion of the tail Phenolics: Two drops of 5% FeCl3 (w/v) was added placed over the light beam of the tail flick appara- to 1 mL of the plant extract. A greenish precipitate tus. A maximum cut-off time of 10 seconds was ob- indicates the presence of phenolics [13]. served to minimize undue tissue damage as a result Cardiac glycosides: (Salkowski Test) − 1 mL of of over exposure of the tail to heat. The reading was the extract was added to 2 mL of chloroform. Anti-inflammatory and analgesic effects of E. tomentosus 283

Subsequently, H2SO4 was carefully added on the in- 90 ner side of the test tube. A reddish-brown color at 80 the interface indicates the presence of a glycone 70 portion of cardiac glycoside [14]. 60 * Tannins: 1 mL of freshly prepared 10% (w/v) 50 ethanolic KOH was added to 1 mL of the extract. A 40 * † dirty white precipitate indicates the presence of 30 † tannins [15]. 20 Flavonoids: 1 mL of 10% (w/v) NaOH was added 10

to 3 mL of the extract. A yellow coloration indi- Changes in paw thickness (%) 0 cates the presence of flavonoids [13]. 135 Saponins: (Frothing test) − In a test tube, 2 mL Time (hr) of extract was shaken vigorously with water for Control EtE (500 mg/kg) 2 minutes and warmed. Froth formation that per- Indomethacin (10 mg/kg) EtE (250 mg/kg) sists upon warming indicates the presence of EtE (1000 mg/kg) saponins [16]. Steroids: Five drops of concentrated H SO Figure 1 Effect of the E.tomentosus extract (EtE) and 2 4 indomethacin on the carrageenan-induced hind paw were added to 1 mL of the extract. Red coloration edema. *p < 0.01 and †p < 0.001 from one-way analysis of indicates the presence of steroid [17]. variance (ANOVA) against control group. 2.9. Statistical analysis 10 The results are reported as mean ± standard error 9 mean (S.E.M.). One-way Analysis of Variance (ANOVA) 8 followed by two-tailed Dunnett’s multiple compar- 7 ison test was used for comparisons. The statistical 6 * 5 † methods were performed using SPSS version 10.0 4 (SPSS Inc., Chicago, IL, USA). A p value of < 0.05 was 3 set as statistical significant. 2

Concentration ( μ g/10 mL) 1 0 3. Results

3.1. Acute toxicity study Control Indomethacin(10 mg/kg) EtE (500 mg/kg)EtE (250 mg/kg) In the acute toxicity study, EtE at a dose of EtE (1000 mg/kg) 5000 mg/kg caused neither visible signs of toxicity Figure 2 Effects of E.tomentosus extract (EtE) and nor mortality. A female rat given single dose EtE indomethacin on peritoneal capillary permeability test. (5000 mg/kg) survived after the observation peri- *p < 0.01 and †p < 0.001 from one-way ANOVA against con- ods, and then two additional female rats were given trol group. EtE (5000 mg/kg) and observed for 14 days. All three rats survived during the observation period. of 1000 mg/kg showed a significant inhibition (n = 6; p < 0.05) in magnitude of swelling 3 and 5 hours fol- 3.2. Carrageenan-induced rat hind lowing carrageenan administration. However, no paw edema test sig nificant changes in carrageenan-induced paw edema were observed at EtE doses of 250 and In the carrageenan-induced edema test, the per- 500 mg/kg throughout the whole experimental pe- centage changes of right hind paw thickness by EtE riod or at 1 hour following the injection of carra- and indomethacin are shown in Figure 1. For the geenan in rats treated with 1000 mg/kg. control group, carrageenan caused localized edema starting 1 hour after injection (increased to 30.08 ± 3.3. Acetic acid-induced peritoneal 7.39%). The swelling increased progressively to capillary dye leakage test 72.62 ± 7.3% at 5 hours after carrageenan injection. Indomethacin (10 mg/kg, p.o.) caused a significant The results of acetic acid-induced peritoneal cap- attenuation in hind paw edema 3 and 5 hours after illary dye leakage are shown in Figure 2. The total injection of carrageenan (n = 6; p < 0.001). As with amount of dye was determined from a standard indomethacin, rats treated with the EtE at a dose curve. EtE significantly reduced the Chicago sky 284 M.F. Yam et al

Table 1 Effect of various treatments on the hot plate test*

Response time (s) (n = 8) Group 15 30 45 60 90

Control 10.81 ± 1.06 10.94 ± 1.45 11.41 ± 1.37 10.63 ± 0.9 11.06 ± 0.89 Morphine (8 mg/kg) 14.30 ± 1.92† 18.14 ± 1.99† 21.95 ± 1.81† 30.10 ± 2.8† 21.95 ± 2.05† EtE (mg/kg) 1000 10.24 ± 0.76 12.08 ± 0.82 11.10 ± 1.60 10.54 ± 1.17 12.23 ± 1.33 500 9.84 ± 0.80 11.46 ± 1.06 9.99 ± 0.97 11.94 ± 0.81 12.11 ± 1.54 250 8.90 ± 0.91 12.00 ± 1.24 10.39 ± 0.87 12.96 ± 0.96 10.86 ± 1.20

*Data presented as mean ± standard error mean; †p < 0.001, evaluated by one-way analysis of variance (ANOVA) against control group. blue from leaking at the dose of 1000 mg/kg (n = 8; < Table 2 Effect of Elephantopus tomentosus extract p 0.01) as compared with the control. Indo- (EtE) and morphine treatments on tail flick* methacin (10 mg/kg) also significantly reduced the dye leakage as compared with the control (n = 8; Group Response time (s) (n = 8) p < 0.001). Control 1.88 ± 0.10 † 3.4. Hot plate and tail flick tests Morphine (8 mg/kg) 8.9 ± 0.56 EtE As shown in Table 1 and Table 2, it can be clearly 1000 1.94 ± 0.14 ± observed that morphine produced a significant in- 500 1.88 0.17 ± crease (p < 0.001) in the response time in the hot 250 2.01 0.26 plate and tail flick experiment, respectively. EtE, *Data presented as mean ± standard error mean; †p < 0.001, in contrast, did not significantly alter the response evaluated by one-way ANOVA against control group. time at all the tested doses.

3.5. Carrageenan-induced rat hind paw Time (hr) hyperalgesia pain threshold test 46 0 −10 Indomethacin at a dose of 10 mg/kg p.o. caused a −20 significant increase in the pain threshold 4 and 6 −30 = hours after carrageenan injection (Figure 3, n 6; −40 p < 0.05 and p < 0.01, respectively). Likewise, EtE −50 * † at a dose of 1000 mg/kg showed a significant in- −60 * crease in pain threshold level at 4 and 6 hours fol- −70 * lowing carrageenan administration (Figure 3, n = 6; −80 Changes of force (%) p < 0.05). However, EtE at doses of 250 and 500 mg/kg −90 did not produce any significant changes in the pain −100 threshold during in the experiment. Control EtE (500 mg/kg) Indomethacin (10 mg/kg) EtE (250 mg/kg) 3.6. Acetic acid-induced writhing in mice EtE (1000 mg/kg) Figure 3 Effect of E.tomentosus extract (EtE) and Figure 4 demonstrates that EtE (1000 mg/kg) and pa- indomethacin on the pain threshold of the carrageenan- racetamol (100 mg/kg) caused an inhibition of the induced hyperalgesia hind paw. *p < 0.05 and †p < 0.01 writhing response induced by acetic acid (n = 6; from one-way ANOVA against control group. p < 0.05 and p < 0.01, respectively). The analgesic ef- fect of EtE on the writhing test was dose-dependent. 3.7. Phytochemical analysis The percentage inhibition of writhing produced by EtE at the doses of 1000, 500 and 250 mg/kg in Preliminary phytochemical analysis of the extract mice were 23.1, 8.4 and 2.5%, respectively. Para- revealed the presence of flavonoids, saponins, cetamol (100 mg/kg) exhibited approximately 28.6% tannins, cardiac glycosides, steroids and phenolic inhibition. compounds (data not shown). Anti-inflammatory and analgesic effects of E. tomentosus 285

45 permeability in mice. This result suggests that EtE 40 may produce an anti-inflammatory action through inhibiting the inflammatory mediators of the acute 35 † * 30 phase of inflammation. In the above acute in- 25 flammatory models, EtE showed anti-inflammatory 20 activity similar to the positive control drug indo- 15 methacin, a NSAID which is known to nonselec- 10 tively inhibit the action of the cyclooxygenase Number of writhing enzymes. These data suggest that EtE has an anti- 5 inflammatory property, probably like indomethacin, 0 acting through the inhibition of the inflammatory mediators of the acute phase of inflammation. Control However, further work needs to be carried out to Paracetamol evaluate the effect of EtE on the enzymatic activ- (100 mg/kg) EtE (500 mg/kg)EtE (250 mg/kg) EtE (1000 mg/kg) ity of cyclooxygenase. Figure 4 Effect of the E.tomentosus extract (EtE) and The analgesic properties of EtE were studied paracetamol on the acetic acid-induced writhing test. using four important laboratory models, namely *p < 0.05 and †p < 0.01 from one-way ANOVA against con- the hot plate latency method, tail flick latency trol group. method, acetic acid-induced writhing method and carrageenan-induced hyperalgesia method. The 4. Discussion hot plate and tail flick methods are sensitive to centrally-acting analgesic drugs [24,25] while car- In the present study, the anti-inflammatory and rageenan-induced hyperalgesia and acetic acid analgesic properties of EtE were investigated. The models typically resemble human clinical pain con- carrageenan-induced edema test was selected be- ditions and are sensitive to peripherally-acting an- cause of its sensitivity in detecting orally active algesic drugs [26,27]. The hot plate and tail flick anti-inflammatory agents, particularly natural prod- tests were undertaken to verify if EtE could show ucts, and as an acceptable and reliable method for any central anti-nociceptive effect. In the hot acute anti-inflammatory studies [18]. This study plate and tail flick tests, EtE hardly produced any demonstrates that EtE is effective in reducing car- significant effect. It did not increase tail flick re- rageenan-induced hind paw edema. EtE shows a sponse and latency of the licking or jumping of dose-dependent reduction in the hind paw edema mice as compared with those obtained for the con- at 5 hours after carrageenan administration. Gen- trol group. It is known that NSAIDs do not usually erally, two different phases of inflammation are in- increase the pain threshold level in normal tissues, volved in carrageenan-induced edema [19,20]. The unlike central anesthetics and narcotics, hence, it first phase (0 − 2.5 hours after injection of carra- can be suggested that EtE has no central analgesic geenan) mainly results from concomitant release of effect. inflammation mediators, namely histamine, seroto- Previous studies have showed that sub-dermal nin and kinins from damaged surrounding tissues. injections of carrageenan into the rat paw caused a The second phase (3 − 6 hours after injection of car- marked hyperalgesia 4 hours post-injection, which rageenan) is mediated by bradykinin, leukotrienes, was mainly due to prostaglandins [28]. The 1000 mg/ and prostaglandins produced by the macrophages kg EtE-treated rats showed a significant increase and sustained by released prostaglandins [18]. Oral in pain threshold level at 4 and 6 hours after carra- administration of EtE (1000 mg/kg) suppressed the geenan injection. The intraperitoneal administra- edematous response 3 hours after carrageenan in- tion of acetic acid induced a stereotypical behavior jection and this effect continued for up to 5 hours. in mice, which is characterized by abdominal con- These results indicate that EtE acts in the second tractions, movements of the body as a whole, twist- phase, which mainly involves arachidonic acid ing of dorsoabdominal muscles and a reduction in metabolites [20−22]. motor activity and coordination [29]. The algesic Vascular permeability was induced by acetic mechanism of abdominal writhing involves the re- acid. Acetic acid causes an increase in prostaglan- lease of arachidonic acid via prostaglandin biosyn- dins, serotonin and histamine release. This in turn thesis and sympathetic nervous system mediators leads to a dilation of the capillary vessels and thus [30−32]. Thus, the results obtained for the writhing an increase in vascular permeability. As a conse- test using acetic acid are similar to those obtained quence, fluid and plasma proteins are extravasated for the edematogenic test using carrageenan, since and lead to edema formation [23]. EtE markedly in- EtE at a dose of 1000 mg/kg significantly inhibited hibited the acetic acid-induced increase in vascular the writhing activity. Therefore, anti-inflammatory 286 M.F. Yam et al substances in EtE may also be involved in peripheral 7. Winter CA, Risley EA, Nus GV. Carrageenan-induced edema analgesic activity [33]. in hind paw of the rat as an assay for anti-inflammatory − Phytochemical analysis also showed that the drug. Proc Soc Exp Biol Med 1962;111:544 7. 8. Whittle BA. The use of changes in capillary permeability in extract contains saponins, cardiac glycosides, phe- mice to distinguish between narcotic and nonnarcotic anal- nolic compounds, tannin, steroid, and flavonoids. gesics. Br J Pharmacol Chemother 1964;22:246−53. 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