Anti-inflammatory activity of Wigandia urens and Acalypha alopecuroides

The anti-inflammatory activity of the chloroform, methanol and aqueous extracts of Wigandia urens and Acalypha alopecuroides were investigated on carrageenan-induced paw edema at doses of 400 mg/kg. The three extracts of W. urens, and the aqueous extract of A. alopecuroides caused significant inhibition of the edema (58.1±6.5% and 63.5±5.4%, respectively). Indomethacin was used as positive control (8 mg/kg), and inhibited edema by 66.3±5.2%. The methanol extract of W. urens and the aqueous extract of A. alopecuroides, at doses of 200 mg/kg, inhibited pellet implantation-induced granuloma formation by 69.4±6.5 and 70.6±6.6%, respectively. These levels of inhibition are higher than those exhibited by naproxen at doses of 50 mg/kg (46.1±7.1%). Both extracts showed activity on adjuvantinduced arthritis in rats, with the best effect being observed after 96 h (82.2±4.6 and 80.6±7.3%, respectively).


INTRODUCTION
Inflammation is a basic response to many injuries and is characterized by redness, warmth, swelling and pain.The compounds normally used to treat these symptoms may induce side-effects, which has lead to the search for natural compounds that could be useful in treating these kinds of disorders.
Wigandia urens (Ruiz and Pav.) HBK (Hidrofilaceae), commonly known as "suelda con suelda", is a perennial that grows to a height of 3.0-3.6m (González- Vazquez-Yañez et al., 2001).This plant is frequently employed in Mexican folk Medicine for the treatment of rheumatic and muscular pain, as well as inflammatory problems.Previous studies have shown that W. urens contains flavornoids (Wollenweber et al., 1996) and phenolic derivatives (Cao et al., 2003).
Here, the anti-inflammatory activity of three extracts of the aerial parts of W. urens and A. alopecuroides is reported in various animal models.

Animals
The male Wistar rats (150-250 g) used for this study were housed at 20°C under a 12 h light/12 h dark cycle.The animals were fed (Purina) and had access to water ad libitum.
All the experiments were performed according to the current guidelines for the care of laboratory animals and the ethical guidelines for the use and care of laboratory animals NOM-062-ZOO-1999.

Plant material
The aerial parts of W. urens were collected in Zirandaro, Guerrero State, Mexico in May, 2003. A. alopecuroides was collected around Fortín de las Flores, Veracruz State, México in September, 2003.Both plants were identified by MS Aurora Chimal of the Department of El Hombre y su Ambiente, Universidad Autónoma Metropolitana-Xochimilco, where voucher specimens (MSP72 and MSP73 respectively) were deposited in the herbarium.

Preparation of the extracts
The shade-dried aerial parts were reduced to a powdery form and 100 g of the powdered sample was refluxed for 4 h with 700 mL of chloroform, methanol or water.The resulting extracts were then filtered.The chloroform and methanol were removed under reduced pressure (yield 5.3 and 7.6%, for W. urens and for A. alopecuroides 4.7 and 6.8%, respectively), and the water was removed by lyophilization (yield of W. urens 10.5 and 10.3% for A. alopecuroides).
A preliminary screening of the methanol extract of W. urens showed a positive FeCl3 reaction for phenolic compounds (Domínguez, 1973), a positive Tortelli-Jaffe and Tschugaeff test for terpenes, a positive boric acid and citric acid test for flavonoids and positive silicotungstic acid and Dragendorff reactions for alkaloids (Domínguez, 1973).The aqueous extract of A. alopecuroides show-ed a positive FeCl3 reaction for phenolic compounds, positive Lieberman-Burchard test for terpenes and show positive reaction for saponins (Domínguez, 1973;Harborne, 1998).

Anti-inflammatory activity
The anti-inflammatory activity of the extracts of W. urens and A. alopecuroides was investigated using the following animal models.

Carrageenan-induced paw edema in rats
Pedal inflammation in male Wistar rats was produced according to the method described by Winter et al. (Winter et al., 1962).Edema in the left hind paw was induced by injection of 0.1 mL of a 1% carrageenan solution into the sub-plantar region.The paw volume of each rat was measured at 1.5, 3.0 and 5.0 h after carrageenan injection with a plethysmometer (Ugo Basile).The drug test groups were treated orally with 50, 100, 200 or 400 mg/kg of the methanol extracts or 200 mg/kg of the chloroform or aqueous extract 1 h before carrageenan injection.The animals in the control group received (p.o.) the vehicle (5% Tween 80).Another group of rats was administered indomethacin (8 mg/kg) as a standard reference.The edema inhibition of each group was calculated according to Olajide et al. (Olajide et al., 2000).
where Ct is the displacement volume at time t after carrageenan administration, and Co is the displacement volume before carrageenan administration.

Cotton pellet-induced granuloma
Rats were anesthetized and a 3.0 mg cotton pellet was placed subcutaneously through a skin incision at the sternum level (Winter and Porter, 1957).Once daily, beginning 30 min postoperatively, groups of 6 rats were dosed p.o. with 1 mL of water and 25 mg/kg of naproxen (as a positive control), the methanol extract of W. urens or the aqueous extract of A. alopecuroides (200 mg/kg) in a 5% Tween 80 solution.The administration was continued for 5 days.On the sixth day, the animals were sacrificed with ether, and the pellet with the accompanying granuloma tissue (diameter 6 mm) and a plug of the same size on the opposite side of the granuloma were excised, dried for 24 h at 60°C and weighed.The difference between the weight of the granuloma and the plug without the cotton pellet weight was considered in the determination of the amount of granulomatous tissue produced.

Adjuvant-induced arthritis
The method used to induce arthritis was described by Mizushima et al. (1972).Briefly, 0.1 mL of Freund's complete adjuvant was injected at the base of the tail of groups of Wistar rats.Six days after adjuvant inoculation, 0.1 mL of 1% carrageenan solution was injected into the sub-plantar region of the left hind paw, and 1 h after that, the group was treated orally with 200 mg/kg of the methanol extract of W. urens or the aqueous extract of A. alopecuroides.At the same time, the control group received p.o. vehicle (5% Tween 80), while the positive control was treated with 8 mg/kg of indomethacin.The degree of pedal edema was determined by measuring the left hind paw volume with a plethysmometer (Ugo Basile).These measurements were made before the adjuvant injection and were repeated again 6 days later at 3 and 5 h (acute phase), and continued for 24 to 96 h after carrageenan injection.The edema volume is expressed as the difference found in the left hind paw compared with the right hind paw.The inhibition of edema was calculated for each animal group in comparison with the control group.

Statistical analysis
Values are expressed as mean ± S.E.M. Treated groups were compared with the controls to statistically assess differences (p < 0.05 was deemed significant) via ANOVA followed by Dunnett's multiple comparison test.

RESULTS
Table 1 shows the inhibitory effect of the chloroform, methanol and aqueous extracts of A. alopecuroides and W. urens on carrageenan-induced paw edema.Both the chloroform and methanol extracts of A. alopecuroides were inactive in this model.However, the edema was markedly inhibited by oral pre-treatment at doses of 400 mg/kg of the methanol or aqueous extracts of W. urens and the aqueous extract of A. alopecuroides (Table 1).
The methanol extract of W. urens and the aqueous extract of A. alopecuroides were also tested at doses of 50, 100, 200 and 400 mg/kg (Table 2).The methanol extract of W. urens showed the strongest activity at doses of 400 mg/kg (53.6%) after 90 min of administration of the flogistic agent.However, the anti-inflammatory activity with different doses of the methanol extract remained for 180 and 300 min after administration, and the effect was similar to that observed with Indomethacin at doses of 8 mg/kg (63.4 and 73.3%, respectively).The aqueous extract of A. alopecuroides had no effect at doses of 50 mg/kg, and the greatest effect was observed at a dose of 400 mg/kg after 90 min of administration (63.6%).This effect was similar at 180 and 300 min (68.1 and 72.2%, respectively).The methanol extract of W. urens at doses of 200 mg/ kg was found to reduce 69.4 ± 6.5% of the weight of cotton pellet-induced granuloma, and the aqueous extract of A. alopecuroides reduced the edema by 70.6 ± 6.6%.Both extracts demonstrated inhibition of edema that was higher than that obtained when naproxen was administered (46.1±7.1%) at doses of 25 mg/kg (Table 3).
After 24 h of oral administration of the aqueous extract of A. alopecuroides at doses of 200 mg/kg, the inhibition was 20.3%, and this effect was increased after 72 h (77.5%), with the greatest inhibition being observed at 96 h (82.2%).Both extracts exhibited an important antiarthritic effect in the chronic phase compared to indomethacin, with an inhibition of 46.9 and 75.5% after 5 and 24 h, respectively.The inhibitory effect diminished after 48 h (58.1%) and 92 h (37.9 %).The results are significant (p < 0.05) compared to the 5% Tween 80-treated animals.

DISCUSSION
In the present study, the anti-inflammatory effects of the methanol extract of W. urens and aqueous extract of A. alopecuroides on acute and chronic inflammatory processes has been established.It is well known that edema originated by the action of carrageenan in the rat paw involves different phases as leukocytes migrate to the injured tissues (Carvalho et al., 1999), alter the neutrophil membrane and produce highly reactive oxygen species, such as superoxide.Neutrophils plays an essential role in acute phlogosis (Galati et al., 2005).The early phase (1-2 h) of this model is mainly mediated by histamine and serotonin release (Olajide et al., 2000).The other phase is due to the release of kinins, prostaglandins, proteases and lysosomes (Alexandre-Moreira et al., 1999).The anti-inflammatory effect of the methanol extract of W. urens and the aqueous extract of A. alope- curoides in rats with carrageenan-induced edema was significant at doses of 200 and 400 mg/kg for the study time (5 h) (Table 2).Cotton pellet-induced granuloma is the typical model used to evaluate the transudative and proliferative components of the chronic inflammatory reaction (Ismail et al., 1997).In this case, the dry weight of the pellets correlates well with amount of granulomatous tissue (Swinglen and Shideman, 1972).The methanol extract of W. urens, the aqueous extract of A. alopecuroides (200 mg/kg) and naproxen (25 mg/kg) are effective at lowering the moist weight of the cotton pellet, and the effect of the extracts was higher than that demonstrated by naproxen (Table 3).This effect correlates with the ability of both extracts in reducing the proliferative phase of granuloma tissue formation (Ojalde et al., 2000).
The methanol extract of W. urens and the aqueous extract of A. alopecuroides also exhibited anti-arthritic effects in the chronic phase (Table 4).This extract decreased the edema induced 48 h after carrageenan treatment (6 days after adjuvant inoculation).The effect lasted up to 72 h and was higher than that observed with indomethacin at these time points.Based upon the results obtained, it is concluded that the methanol extract of W. urens and the aqueous extract of A. alopecuroides have activity in both the acute and chronic phases of inflammation, thereby supporting the use of W. urens and A. alopecuroides in various ailments in Mexican folk medicine.Detailed studies are in progress to identify the structures of the active principle(s) present in the methanol extract of W. urens and aqueous extract of A. alopecuroides.

Table 1 .
Effect of chloroform, methanol and aqueous extracts of W. urens and A. alopecuroides at doses of 400 mg/Kg on paw edema of rats induced with carrageenan.Results are the mean of at least 5 determinations ± standard error of increase in paw volume.Values indicate percent reduction in paw volume compared with the control group treated only with carrageenan.n =7, Dunnett's test * p< 0.05.N.E.= No effect.

Table 2 .
Effect of aqueous extract of A. alopecuroides and methanol extract of W. urens at doses of 50, 100, 200 and 400 mg/Kg on paw edema of rats induced with carrageenan.
Results are the mean of at least 5 determinations ± standard error of increase in paw volume.Values indicate percent reduction in paw volume compared with the control group treated only with carrageenan.n=7, Dunnett's test * p< 0.05.N.E.= No effect.

Table 3 .
Effect of the methanol extract of W. urens and aqueous extract of A. alopecuroides (consecutive for 5 days) on the weight of granuloma in rats.

Table 4 .
Effect of the aqueous extract of A. alopecuroides and methanol extract of W. urens on rat treatment with Freund's adjuvant and paw edema induced with carragenan.
Results are the mean of at least 5 determinations ± standard error of increase in paw volume.Values indicate percent reduction in paw volume compared with the control group treated only with carrageenan.n=7, Dunnett's test * p< 0.05.N.E.= No effect.