Anti-inflammatory and Analgesic Effects of Elephantopus tomentosus Ethanolic Extract

Article Outline

• Abstract
• 1.. Introduction
• 2.. Materials and Methods
  • 2.1.. Materials
  • 2.2.. Preparation of plant extract
  • 2.3.. Animals
  • 2.4.. Acute toxicity
  • 2.5.. Carrageenan-induced rat hind paw edema
  • 2.6.. Acetic acid-induced peritoneal capillary dye leakage test
  • 2.7.. Analgesic studies
    • 2.7.1.. Hot plate test
    • 2.7.2.. Tail flick test
    • 2.7.3.. Carrageenan-induced rat hind paw hyperalgesia pain threshold test method
    • 2.7.4.. Acetic acid-induced writhing test
  • 2.8.. Phytochemical analysis
  • 2.9.. Statistical analysis
• 3.. Results
  • 3.1.. Acute toxicity study
  • 3.2.. Carrageenan-induced rat hind paw edema test
  • 3.3.. Acetic acid-induced peritoneal capillary dye leakage test
  • 3.4.. Hot plate and tail flick tests
  • 3.5.. Carrageenan-induced rat hind paw hyperalgesia pain threshold test
  • 3.6.. Acetic acid-induced writhing in mice
  • 3.7.. Phytochemical analysis
• 4.. Discussion
• References
• Copyright

Abstract 

Elephantopus tomentosus is widely used in Asia, especially in Malaysia, for the treatment of pain and inflammation. In the present study, the analgesic and anti-inflammatory effects of a 95% ethanol extract of E. tomentosus were investigated in different experimental models. In the anti-inflammation study, 1000 mg/kg of extract significantly reduced carrageenan-induced hind paw edema (p < 0.05) and inhibited abdominal permeability compared with control (p < 0.01). The analgesic 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 significantly (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.

Key Words:  analgesic effect , anti-inflammatory , Elephantopus tomentosus

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1. Introduction 

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

Members of the Elephantopus genus are widely used in Southeast Asian folk medicine. Elephantopus spp is believed to have anti-pyretic, anti-inflammatory and emollient activities [5]. In Malaysia, E. tomentosus (Et) is taken internally as a diuretic, febrifuge, analgesic, anti-helminthic and anti-inflammatory agent. Et is also applied externally as a poultice for abdominal pains. However, very few scientific studies on Et have been conducted. Recently, Et was found to exhibit antioxidant properties and a liver protective effect on carbon tetrachloride-induced liver toxicity [6]. With this background, the present study aimed to investigate the analgesic and anti-inflammatory effects of E.tomentosus ethanol extract (EtE) using different pharmacological experimental models in an attempt to validate its ethnomedical use.

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2. Materials and Methods 

2.1. Materials 

Carrageenan type IV (Carrageenan), sodium hydroxide (NaOH), ferric chloride (FeCl₃), potassium chlo-ride, sodium chloride (NaCl), potassium hydroxide (KOH), Chicago sky blue, and indomethacin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carboxymethylcellulose (CMC) was supplied from British Drug House (Poole, England). Ethanol was purchased from Riedel-de Haën (Seelze, Germany). Mayer reagent was obtained from Fluka Chemie GmbH (Buchs, Switzerland). Glacial acetic acid, hydro-chloric acid (HCl) and sulfuric acid (H₂SO₄) were obtained from Merck (Darmstadt, Germany). Morphine sulfate was purchased from Remedi Pharmaceutical (Selangor, Malaysia).

2.2. Preparation of plant extract 

The plant was obtained from Permatang Damar Laut, Penang, Malaysia. The plant was sent to the Herbarium, School of Biological Sciences, Universiti Sains Malaysia for identification. The herbarium voucher number is 10832. After identification, the plant was dried in an oven (Memmert, Germany) at 45°C and ground into powder. The powered plant material (250 g) was put into a thimble and extracted with 5 L of 95% ethanol in a soxhlet extractor for 3 days. The extract was concentrated using Büchi RE121 rotary evaporator (Büchi Labortechnik AG, Switzerland) and subsequently dried in a Hetovac VR-1 freeze dryer (Heto Lab. Equipment AS, Denmark). The yield (EtE) obtained was found to be 14% (w/w) of the dried plant.

2.3. Animals 

Male and female Sprague Dawley (SD) rats (age, 8-12 weeks; weight, 150-200 g) and Institute Charles River strain (ICR) mice (age, 3-4 weeks; weight, 25-32 g) were used for the experiments. The animals were kept under stan dard conditions (26–30°C) with free access to food (normal laboratory chow, Gold Coin Feed Mills Sdn Bhd, Malaysia) and tap water. The animals were fasted for 6 hours prior to the experiments and acclimatized to laboratory conditions for 7 days before commencement of experiments. All the experimental procedures on animals were carried out in accordance with the regulations and guidelines of the Animal Ethics Committee, Universiti Sains Malaysia.

2.4. Acute toxicity 

Organisation for Economic Co-operation and Development guidelines for testing of chemicals were adhered. Healthy female SD rats were subjected to tests according to the acute oral toxicity upand-down procedure (limit test). All experimen tal animals were maintained under close observation for 14 days.

2.5. Carrageenan-induced rat hind paw edema 

The anti-inflammatory activity was evaluated according to the method described by Winter et al (1962) [7]. A total of 30 male rats were used in the experiment. They were divided into five groups of six rats each. Carrageenan was freshly prepared as 1% (w/v) suspension in sterile 0.9% NaCl before the experiment. A volume of 0.1 mL of carrageenan was injected into the plantar tissue of the rat right hind paw. EtE (250, 500, and 1000 mg/kg) and indomethacin (10 mg/kg) were administered by oral gavage 1 hour before carrageenan injection. The animals in the control group received 1% of CMC in distilled water. The footpad thickness was mea sured simply by placing the unanesthetized animal foot between the anvil and spindle of peacock dial thickness gauge of a micrometer (Ozaki Ltd., Japan) before injection and 1, 3 and 5 hours after the injection of carrageenan. The percentage in thickness changes of the hind paw was calculated according to the following formula:

C_t is the thickness of hind paw at t hour. C₀ is the thickness of hind paw before carrageenan was injected.

2.6. Acetic acid-induced peritoneal capillary dye leakage test 

The capillary permeability or dye leakage test was performed by modifying a previously reported method (Whittle, 1964) [8]. The ICR mice (four males and four females in each group) were given EtE (250, 500 and 1000 mg/kg) and indomethacin (10 mg/kg). The control group was orally given 1% CMC in distilled water. After 25 minutes, each animal was given an intravenous injection of 0.1 mL of 4% solution of Chicago sky blue in normal saline. After 30 minutes, 0.2 mL of 0.25% (v/v) glacial acetic acid solution in normal saline was injected intraperitoneally. After an additional 20 minutes, the mice were killed by cervical dislocation, and the visceral organs were subsequently exposed. A period of 1 minute was allowed for blood to drain away from the abdominal wall and jugular vein. The animals were then held by a flap of the abdominal wall and the exposed visceral organs were continuously irrigated with distilled water in a petri dish. The combined washings were filtered through glass wool and filled a 10 mL volumetric flask. Then, 0.1 mL of 0.1 N NaOH was subsequently added to deproteinize the filtrate. The absorbance was measured using a Hitachi U-2000 spectrophotometer at 590 nm (Hitachi, Japan). The total concentration of Chicago sky blue was determined as m g/10 mL from a standard calibration curve.

2.7. Analgesic studies 

The mice were placed on an Ugo Basile 7280 hot plate (Ugo Basile, Italy), which was maintained at 55 ± 0.5°C. The time between placement and licking of the paws or jumping was recorded as latency second. The latency was recorded at 15, 30, 45, 60 and 90 minutes after administration of EtE (250, 500, and 1000 mg/kg, p.o.), morphine sulfate (8 mg/kg, s.c.) and vehicle (10 mL/kg of 1% CMC, p.o.) [2]. Mor phine sulfate was used as a reference standard.

The nociception was assessed using an Ugo Basile 7360 tail flick apparatus (Ugo Basile, Italy). Briefly, the tail flick test response was done by measuring the time taken to withdraw the tail from the heat source [2]. Each mouse was encased in a small aluminum chamber with the middle portion of the tail placed over the light beam of the tail flick apparatus. A maximum cut-off time of 10 seconds was observed to minimize undue tissue damage as a result of over exposure of the tail to heat. The reading was taken 1 hour after administration of EtE (250, 500, 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 purpose of calculation, an animal not responding by the cut-off time of 10 seconds was arbitrarily assigned a response time of 10 seconds.

The hyperalgesia pain threshold of the inflamed rat's right hind paw was investigated by subjecting the hind paw to a constant force following the method of Winter and Flataker [9]. The various treatments [EtE (250, 500, and 1000 mg/kg, p.o.), indomethacin (10 mg/kg, p.o.) and 1% CMC (10 mL/kg, p.o.)] were respectively given 2 hours after a sub-plantar injection of 0.1 mL of 1% carrageenan. Pain threshold was measured mechanically using a Letica Analgesy-meter LI7306 (Letica Scientific Instruments, Spain) before the injection of carrageenan and then at 4 and 6 hours post-injection. The pain threshold (g) was measured when the rat withdrew its hind paw, struggled or vocalized [10]. The percentage change of pain threshold was calculated according to the following formula:

P_t is the pain threshold at t hour and P₀ is the pain threshold before carrageenan was injected.

The method of Koster et al [11] was used in this study. EtE (250, 500 and 1000 mg/kg), paracetamol (100 mg/kg) and 1% CMC were orally administered to respective groups of mice. One hour later, each mouse was injected with 0.6% (v/v) acetic acid (10 mL/kg, i.p.). The number of writhings during the 15 minutes was recorded.

2.8. Phytochemical analysis 

Preliminary phytochemical analysis of the extract was performed using the following method:

Alkaloids: 1 mL of 1% HCl (v/v) was added to 3 mL of plant extract in a test tube. The mixture was heated for 20 minutes, cooled and filtered. Two drops of Mayer's reagent was added to 1 mL of the filtrate. A creamy precipitate indicates the presence of alkaloids in the extract [12].

Phenolics: Two drops of 5% FeCl₃ (w/v) was added to 1 mL of the plant extract. A greenish precipitate indicates the presence of phenolics [13].

Cardiac glycosides: (Salkowski Test) −1 mL of the extract was added to 2 mL of chloroform. Subsequently, H₂SO₄ was carefully added on the inner side of the test tube. A reddish-brown color at the interface indicates the presence of a glycone portion of cardiac glycoside [14].

Tannins: 1 mL of freshly prepared 10% (w/v) ethanolic KOH was added to 1 mL of the extract. A dirty white precipitate indicates the presence of tannins [15].

Flavonoids: 1 mL of 10% (w/v) NaOH was added to 3 mL of the extract. A yellow coloration indicates the presence of flavonoids [13].

Saponins: (Frothing test) – In a test tube, 2 mL of extract was shaken vigorously with water for 2 minutes and warmed. Froth formation that persists upon warming indicates the presence of saponins [16].

Steroids: Five drops of concentrated H₂SO₄ were added to 1 mL of the extract. Red coloration indicates the presence of steroid [17].

2.9. Statistical analysis 

The results are reported as mean ± standard error mean (S.E.M.). One-way Analysis of Variance (ANOVA) followed by two-tailed Dunnett's multiple comparison test was used for comparisons. The statistical methods were performed using SPSS version 10.0 (SPSS Inc., Chicago, IL, USA). A p value of < 0.05 was set as statistical significant.

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3. Results 

3.1. Acute toxicity study 

In the acute toxicity study, EtE at a dose of 5000 mg/kg caused neither visible signs of toxicity nor mortality. A female rat given single dose EtE (5000 mg/kg) survived after the observation periods, and then two additional female rats were given EtE (5000 mg/kg) and observed for 14 days. All three rats survived during the observation period.

3.2. Carrageenan-induced rat hind paw edema test 

In the carrageenan-induced edema test, the percentage changes of right hind paw thickness by EtE and indomethacin are shown in Figure 1. For the control group, carrageenan caused localized edema starting 1 hour after injection (increased to 30.08 ± 7.39%). The swelling increased progressively to 72.62 ± 7.3% at 5 hours after carrageenan injection. Indomethacin (10 mg/kg, p.o.) caused a significant attenuation in hind paw edema 3 and 5 hours after injection of carrageenan (n = 6; p < 0.001). As with indomethacin, rats treated with the EtE at a dose of 1000 mg/kg showed a significant inhibition (n = 6; p < 0.05) in magnitude of swelling 3 and 5 hours following carrageenan administration. However, no sig nificant changes in carrageenan-induced paw edema were observed at EtE doses of 250 and 500 mg/kg throughout the whole experimental period or at 1 hour following the injection of carrageenan in rats treated with 1000 mg/kg.

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• Figure 1. 

  Effect of the E.tomentosus extract (EtE) and indomethacin on the carrageenan-induced hind paw edema. *p < 0.01 and †p < 0.001 from one-way analysis of variance (ANOVA) against control group.

3.3. Acetic acid-induced peritoneal capillary dye leakage test 

The results of acetic acid-induced peritoneal capillary dye leakage are shown in Figure 2. The total amount of dye was determined from a standard curve. EtE significantly reduced the Chicago sky blue from leaking at the dose of 1000 mg/kg (n = 8; p < 0.01) as compared with the control. Indomethacin (10 mg/kg) also significantly reduced the dye leakage as compared with the control (n = 8; p < 0.001).

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• Figure 2. 

  Effects of E.tomentosus extract (EtE) and indomethacin on peritoneal capillary permeability test. *p < 0.01 and †p < 0.001 from one-way ANOVA against control group.

3.4. Hot plate and tail flick tests 

As shown in Table 1 and Table 2, it can be clearly observed that morphine produced a significant increase (p < 0.001) in the response time in the hot plate and tail flick experiment, respectively. EtE, in contrast, did not significantly alter the response time at all the tested doses.

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

Table 2. Effect of Elephantopus tomentosus extract (EtE) and morphine treatments on tail flick^*

3.5. Carrageenan-induced rat hind paw hyperalgesia pain threshold test 

Indomethacin at a dose of 10 mg/kg p.o. caused a significant increase in the pain threshold 4 and 6 hours after carrageenan injection (Figure 3, n = 6; p < 0.05 and p < 0.01, respectively). Likewise, EtE at a dose of 1000 mg/kg showed a significant increase in pain threshold level at 4 and 6 hours following carrageenan administration (Figure 3, n = 6; p < 0.05). However, EtE at doses of 250 and 500 mg/kg did not produce any significant changes in the pain threshold during in the experiment.

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• Figure 3. 

  Effect of E.tomentosus extract (EtE) and indomethacin on the pain threshold of the carrageenan-induced hyperalgesia hind paw. *p < 0.05 and †p < 0.01 from one-way ANOVA against control group.

3.6. Acetic acid-induced writhing in mice 

Figure 4 demonstrates that EtE (1000 mg/kg) and paracetamol (100 mg/kg) caused an inhibition of the writhing response induced by acetic acid (n = 6; p < 0.05 and p < 0.01, respectively). The analgesic effect of EtE on the writhing test was dose-dependent. The percentage inhibition of writhing produced by EtE at the doses of 1000, 500 and 250 mg/kg in mice were 23.1, 8.4 and 2.5%, respectively. Paracetamol (100 mg/kg) exhibited approximately 28.6% inhibition.

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• Figure 4. 

  Effect of the E.tomentosus extract (EtE) and paracetamol on the acetic acid-induced writhing test. *p < 0.05 and †p < 0.01 from one-way ANOVA against control group.

3.7. Phytochemical analysis 

Preliminary phytochemical analysis of the extract revealed the presence of flavonoids, saponins, tannins, cardiac glycosides, steroids and phenolic compounds (data not shown).

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4. Discussion 

In the present study, the anti-inflammatory and analgesic properties of EtE were investigated. The carrageenan-induced edema test was selected because of its sensitivity in detecting orally active anti-inflammatory agents, particularly natural products, and as an acceptable and reliable method for acute anti-inflammatory studies [18]. This study demonstrates that EtE is effective in reducing carrageenan-induced hind paw edema. EtE shows a dose-dependent reduction in the hind paw edema at 5 hours after carrageenan administration. Generally, two different phases of inflammation are involved in carrageenan-induced edema [19, 20]. The first phase (0-2.5 hours after injection of carrageenan) mainly results from concomitant release of inflammation mediators, namely histamine, serotonin and kinins from damaged surrounding tissues. The second phase (3-6 hours after injection of carrageenan) is mediated by bradykinin, leukotrienes, and prostaglandins produced by the macrophages and sustained by released prostaglandins [18]. Oral administration of EtE (1000 mg/kg) suppressed the edematous response 3 hours after carrageenan injection and this effect continued for up to 5 hours. These results indicate that EtE acts in the second phase, which mainly involves arachidonic acid metabolites [20, 21, 22].

Vascular permeability was induced by acetic acid. Acetic acid causes an increase in prostaglandins, serotonin and histamine release. This in turn leads to a dilation of the capillary vessels and thus an increase in vascular permeability. As a consequence, fluid and plasma proteins are extravasated and lead to edema formation [23]. EtE markedly inhibited the acetic acid-induced increase in vascular permeability in mice. This result suggests that EtE may produce an anti-inflammatory action through inhibiting the inflammatory mediators of the acute phase of inflammation. In the above acute inflammatory models, EtE showed anti-inflammatory activity similar to the positive control drug indomethacin, a NSAID which is known to nonselectively inhibit the action of the cyclooxygenase enzymes. These data suggest that EtE has an anti-inflammatory property, probably like indomethacin, acting through the inhibition of the inflammatory mediators of the acute phase of inflammation. However, further work needs to be carried out to evaluate the effect of EtE on the enzymatic activity of cyclooxygenase.

The analgesic properties of EtE were studied using four important laboratory models, namely the hot plate latency method, tail flick latency method, acetic acid-induced writhing method and carrageenan-induced hyperalgesia method. The hot plate and tail flick methods are sensitive to centrally-acting analgesic drugs [24, 25] while carrageenan-induced hyperalgesia and acetic acid models typically resemble human clinical pain conditions and are sensitive to peripherally-acting analgesic drugs [26, 27]. The hot plate and tail flick tests were undertaken to verify if EtE could show any central anti-nociceptive effect. In the hot plate and tail flick tests, EtE hardly produced any significant effect. It did not increase tail flick response and latency of the licking or jumping of mice as compared with those obtained for the control group. It is known that NSAIDs do not usually increase the pain threshold level in normal tissues, unlike central anesthetics and narcotics, hence, it can be suggested that EtE has no central analgesic effect.

Previous studies have showed that sub-dermal injections of carrageenan into the rat paw caused a marked hyperalgesia 4 hours post-injection, which was mainly due to prostaglandins [28]. The 1000 mg/kg EtE-treated rats showed a significant increase in pain threshold level at 4 and 6 hours after carrageenan injection. The intraperitoneal administration of acetic acid induced a stereotypical behavior in mice, which is characterized by abdominal contractions, movements of the body as a whole, twisting of dorsoabdominal muscles and a reduction in motor activity and coordination [29]. The algesic mechanism of abdominal writhing involves the release of arachidonic acid via prostaglandin biosynthesis and sympathetic nervous system mediators [30, 31, 32]. Thus, the results obtained for the writhing test using acetic acid are similar to those obtained for the edematogenic test using carrageenan, since EtE at a dose of 1000 mg/kg significantly inhibited the writhing activity. Therefore, anti-inflammatory substances in EtE may also be involved in peripheral analgesic activity [33].

Phytochemical analysis also showed that the extract contains saponins, cardiac glycosides, phenolic compounds, tannin, steroid, and flavonoids. Flavonoids, saponins, tannins, phenolic compounds, and glycosides have all been associated with various degrees of anti-inflammatory and analgesic activities [33, 34, 35, 36, 37]. Therefore, the anti-inflammatory and analgesic effects observed in this study are perhaps due to the activity(s) of one or a combination of some of the identified classes of compounds. The methods used for the identification of phytochemical constituents are preliminary in nature. Therefore, further studies are required to identify the presence of these constituents and their relative concentration in the leaves of the plant.

Based on the classification of Loomis and Hayes [38], substances with an LD₅₀ between 500 and 5000 mg/kg body weight are regarded as being slightly toxic, while those between 5000 and 15,000 mg/kg body weight are regarded as practically non-toxic. Results presented herein show that EtE has an LD₅₀ value of more than 5000 mg/kg p.o., suggesting that EtE is practically non-toxic.

In conclusion, the present study indicates that EtE has both anti-inflammatory and analgesic effects, similar to those observed with indomethacin. The mechanism of action of EtE might be associated with the inhibition of prostaglandin synthesis as observed for most NSAIDs. Further investigation on how EtE interferes with the mechanism of prostaglandin synthesis is needed. Our study shows that EtE has the potential to be developed as a new product for pain and inflammation management.

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References 

1. Sosa S , Balick MJ , Arvigo R , Esposito RG , Pizza C , Altinier G , et al.   Screening of the topical anti-inflammatory activity of some Central American plants . J Ethnopharmacol . 2002;81:211–215
   • View In Article
   • MEDLINE
   • CrossRef
2. Yam MF , Asmawi MZ , Basir R . An investigation of the antiinflammatory and analgesic effects of Orthosiphon stamineus leaf extract . J Med Food . 2008;11:362–368
   • View In Article
   • CrossRef
3. Andrade SF , Lemos M , Comunello E , Noldin VF , Filho VC , Niero R . Evaluation of the antiulcerogenic activity of Maytenus robusta (Celastraceae) in different experimental ulcer models . J Ethnopharmacol . 2007;113:252–257
   • View In Article
   • CrossRef
4. Gooch K , Culleton BF , Manns BJ , Zhang J , Alfonso H , Tonelli M , et al.   NSAID use and progression of chronic kidney disease . Am J Med . 2007;120: 280e1–280e7
   • View In Article
5. Jaganath IB , Ng LT . In: Herbs the green pharmacy of Malaysia . Kuala Lumpur: Vinpress; 2000;p. 76–77
   • View In Article
6. Yam MF , Basir R , Asmawi MZ , Rosdiah  , Ahmad M , Akowuah GA . Antioxidant and hepatoprotective activities of Elephantopus tomentosus ethanol extract . Pharm Biol . 2008;46:199–206
   • View In Article
7. Winter CA , Risley EA , Nus GV . Carrageenan-induced edema in hind paw of the rat as an assay for anti-inflammatory drug . Proc Soc Exp Biol Med . 1962;111:544–547
   • View In Article
   • MEDLINE
8. Whittle BA . The use of changes in capillary permeability in mice to distinguish between narcotic and nonnarcotic analgesics . Br J Pharmacol Chemother . 1964;22:246–253
   • View In Article
   • MEDLINE
9. Winter CA , Flataker L . Nociceptive thresholds as affected by parenteral administration of irritants and of various antinociceptive drugs . J Pharmacol Exp Ther . 1965;148:373–379
   • View In Article
   • MEDLINE
10. Iwalewa EO , Iwalewa OJ , Adeboye JO . Analgesic, antipyretic, anti-inflammatory effects of methanol, chloroform and ether extracts of Vernonia cinerea less leaf . J Ethnopharmacol . 2003;86:229–234
    • View In Article
    • MEDLINE
    • CrossRef
11. Koster R , Anderson M , DeBeer EJ . Acetic acid for analgesic screening . Fed Proc . 1959;18:412
    • View In Article
12. Harborne JB . In: Phytochemical methods: A guide to modern techniques of plant Analysis . London: Chapman & Hall; 1973;p. 279
    • View In Article
13. Awe IS , Sodipo OA . Purification of saponins of root of Bhlighiasapida Koenig-Holl . Nig J Biochem Mol Biol . 2001;16:s2014
    • View In Article
14. Sofowora A . In: Medicinal Plants and Traditional Medicine in Africa . 2nd ed.. Nigeria: Spectrum Books Ltd; 1993;p. 134–156
    • View In Article
15. Odebiyi A , Sofowora AE . Phytochemical screening of Nigerian medicinal plants (III) . Lloydia . 1978;41:234–246
    • View In Article
    • MEDLINE
16. Owoyele OV , Adebukola OM , Funmilayo AA , Soladoye OA . Anti-inflammatory activities of ethanolic extract of Carica papaya leaves . Inflammopharm . 2008;16:168–173
    • View In Article
17. Trease GE , Evans WC . A textbook of Pharmacognosy . 13th ed.. London: Bailliere-Tyndall Ltd.; 1989;
    • View In Article
18. Brito ARMS , Antonio MA . Oral anti-inflammatory and antiulcerogenic activities of a hydroalcoholic extract and partitioned fractions of Turnera ulmifolia (Turneraceae) . J Ethnopharmacol . 1998;61:215–228
    • View In Article
    • MEDLINE
    • CrossRef
19. Di Rosa M . Review. Biological properties of carrageenan . J Pharm Pharmacol . 1972;24:89–102
    • View In Article
    • MEDLINE
    • CrossRef
20. Di Rosa M , Giroud JP , Willoughby DA . Studies of the acute inflammatory response induced in rats in different sites by carrageenan and turpentine . J Pathol . 1971;104:15–29
    • View In Article
    • MEDLINE
    • CrossRef
21. Crunkhon P , Meacock SER . Mediators of the inflammation induced in the rat paw by carrageenan . Brit J Pharmacol . 1971;42:392–402
    • View In Article
22. Mossa JS , Rafatullah S , Galai AM , Al-Yahya MA . Pharmacological studies of Rhus retinorrhaea . Int J Pharmacog . 1995;33:242–246
    • View In Article
23. Kang HS , Lee JY , Kim CJ . Anti-inflammatory activity of arctigenin from Forsythiae Fructus . J Ethnopharm . 2008;116:305–311
    • View In Article
24. Prado WA , Tonussi CR , Rego EM , Corrado AP . Antinociception induced by intraperitoneal injection of gentamicin in rats and mice . Pain . 1990;4:365–371
    • View In Article
25. Gupta M , Mazumder UK , Kumar RS , Gomathi P , Rajeshwar Y , Kakoti BB , et al.   Anti-inflammatory, analgesic and antipyretic effects of methanol extract from Bauhinia racemosa stem bark in animal models . J Ethnopharmacol . 2005;98:267–273
    • View In Article
    • MEDLINE
    • CrossRef
26. Ferreira SH , Vane SR . New aspects on the mode of action of non-steroid anti-inflammatory drugs . Ann Rev Pharmacol . 1974;14:57–73
    • View In Article
27. Perianayagam JB , Sharma SK , Joseph A , Christina AJM . Evaluation of anti-pyretic and analgesic activity of Emblica officinalis Gaertn . J Ethnopharmacol . 2004;95:83–85
    • View In Article
    • MEDLINE
    • CrossRef
28. Ferreira SH . Prostaglandins . In: Chemical messengers of the inflammatory process . Amsterdam: Elsevier/North-Holland Biomedical Press; 1979;p. 113–151
    • View In Article
29. Bars D , Gozariu M , Cadden SW . Animals models of nociception . Pharmacol Rev . 2001;53:597–652
    • View In Article
    • MEDLINE
30. Koo HJ , Lim KH , Jung HJ , Park EH . Anti-inflammatory evaluation of gardenia extract, geniposide and genipin . J Ethnopharmacol . 2006;103:496–500
    • View In Article
    • MEDLINE
    • CrossRef
31. Hokanson GC . Acetic acid for analgesic screening . J Nat Prod . 1978;41:497–498
    • View In Article
32. Duarte JDG , Nakamura M , Ferreira SH . Participation of the sympathetic system in acetic acid induced writhing in mice . Braz J Med Biol Res . 1988;21:341–343
    • View In Article
    • MEDLINE
33. Garcia-Leme J , Nakamura L , Leite MP , Rochae Silva M . Pharmacological analysis of the acute inflammation process induced in rat's paw by local injection of carrageenan and heating . Brit J Pharmacol . 1973;64:91–98
    • View In Article
34. Hosseinzadeh H , Younesi HM . Antinociceptive and antiinflammatory effects of Crocus sativus L. stigma and petal extracts in mice . BMC Pharmacol . 2002;2:7–16
    • View In Article
    • MEDLINE
    • CrossRef
35. Wang JR , Zhou H , Jiang ZH , Wong YF , Liu L . In vivo antiinflammatory and analgesic activities of a purified saponin fraction derived from the root of Ilex pubescens . Biol Pharm Bull . 2008;31:643–650
    • View In Article
    • CrossRef
36. Thirugnanasambantham P , Viswanathan S , Ramaswamy S , Krishnamurty V , Mythirayee C , Kameswaran L . Analgesic activity of certain flavone derivatives: a structure-activity study . Clinical Exp Pharm Physiol . 2007;20:59–63
    • View In Article
37. Viana GSB , Bandeira MAM , Moura LC , Souza-Filho MVP , Matos FJA , Ribeiro RA . Analgesic and antiinflammatory effects of the tannin fraction from Myracrodruon urundeuva Fr. All . Phytother Res . 1998;11:118–122
    • View In Article
    • CrossRef
38. Loomis TA , Hayes AW . Loomis's Essentials of Toxicology . CA: Academic Press; 1996;
    • View In Article