Anti-inflammatory Properties of Entada abyssinica. Leaves

Abstract

The effects of the defatted methanol extract of Entada abyssinica. Steud. ex A. Rich leaves on some models of inflammation were investigated. The analgesic property of the plant extract was also tested on acetic acid–induced writhing, as well as formalin-induced paw licking, in mice. The antipyretic effect was evaluated using yeast-induced hyperpyrexia in mice. At doses of 50–200 mg/kg, the extract produced significant (p < 0.05) inhibition of leukocyte migration after intraperitoneal injection of carrageenan in rats. A topical anti-inflammatory effect was produced by 20 mg/ear of the extract, as demonstrated by inhibition of croton oil–induced ear edema in mice. The analgesic property of the plant extract was observed by inhibition of acetic acid–induced writhing and paw licking induced by formalin in mice. The extract, however, exhibited no antipyretic activity. This study further established the anti-inflammatory activity of E. abyssinica., in addition to its analgesic effect.

Keywords:

• Analgesic
• anti-inflammatory
• Entada abyssinica
• leukocyte migration
• topical inflammation

Introduction

Entada abyssinica. Steud. ex A. Rich (Mimosaceae) is a tree that is found throughout tropical Africa. The plant has been used for the treatment of bronchitis, coughs, and to alleviate arthritic pains (Kokwaro, 1976). The juice is employed as an instillation for eye inflammation (Watt & Breyer-Brandwijk, 1962; Iwu, 1993).

Earlier, we reported the anti-inflammatory properties of a methanol extract of Entada abyssinica. against carrageenan-induced rat paw edema, cotton pellet granuloma formation in rats, and acetic acid–induced increased vascular permeability in mice (Olajide & Alada, 2001). Here, we report further the anti-inflammatory activities of the plant extract in other models of inflammation. The analgesic and antipyretic effects of the plant were also tested in mice.

Materials and Methods

Plant materials

Entada abyssinica. leaves were collected in Abeokuta, Ogun State, Nigeria, in June 1999 and authenticated in the Herbarium, Forestry Research Institute of Nigeria, Ibadan, Nigeria, where voucher specimens were deposited and assigned a voucher specimen number (FHI 106504). Shade-dried, powdered material was first defatted with n.-hexane for 8 h in a Soxhlet apparatus and then extracted with methanol for 10 h. Solvent removal afforded the solid extract (yield: 8.6%). The extract was dissolved in 0.9% saline for pharmacological tests.

Animals

Male Swiss mice (20–25 g) and male Wistar rats (180–210 g) were used. The animals were bred and housed under normal laboratory conditions of humidity and temperature and light (12 h day:12 h night). They were allowed free access to drinking water and animal pellets. The Principles of Laboratory Animal Care (NIH publication no. 85–23) guidelines and procedures were used in this study (NIH, 1985).

Carrageenan-induced leukocyte migration

This was carried out as earlier described (Olajide et al., 2004), with slight modifications. The extract (50, 100, or 200 mg/kg) was administered orally to rats in different groups. Rats in the reference group received indomethacin (5 mg/kg) orally, while control animals received only saline at a dose of 10 ml/kg. After 1 h, all the animals were injected i.p. with 300 μg/ml carrageenan. Four hours later, the rats were sacrificed and the peritoneal cavities washed with 10 ml phosphate-buffered saline, containing 5 units/ml of heparin and 3% albumin. Total and differential cell counts in the lavage fluid were then performed.

Croton oil–induced ear edema in mice

This experiment was carried out as previously described (Olajide et al., 2000). Mice were anesthetized with ketamine, and a total of 15 µl of an acetone solution containing 75 µg of croton oil and extract at 10, 20, or 40 mg/ear was applied to the inner surface of the right ear of each mouse (surface, about 1 cm²). The left ear was left untreated. Control animals received only the irritant, and indomethacin (100 µg/ear) served as the reference. The animals were sacrificed 6 h later and a plug (6-mm diameter) was removed from both the treated and the untreated ears. The difference in weight between the two plugs was taken as a measure of edematous response.

Acetic acid–induced writhing in mice

This was carried out using the method of Koster et al. (1959). The extract at doses of 50–200 mg/kg was orally administered to mice. One hour later, the mice were injected i.p. with 0.2 ml (3% v/v) acetic acid solution. The number of writhings occurring between 5 and 15 min after acetic acid injection was recorded. The response of the extract-treated animals was compared with that of animals that received indomethacin (5 mg/kg, p.o.) as well as with controls (10 ml/kg saline).

Formalin-induced paw licking in mice

The method of Hunskaar and Hole (1997) was used. A 20 µl solution of 1% formalin was injected into the dorsal surface of the left hind paw of mice after 50, 100, or 200 mg/kg of extract was orally administered to the animals. Control animals received 10 ml/kg saline, and 5 mg/kg indomethacin was administered orally to animals in the reference group. Mice were observed in a chamber; time spent licking the injected paw (licking time) was recorded. Animals were observed for the first 5 min postformalin (early phase), and for 10 min starting at the 20th min postformalin (late phase).

Yeast-induced hyperpyrexia in mice

The method of Loux et al. (1972) was used. Mice were rendered hyperthermic by s.c. injection of 15% aqueous yeast suspension at a dose of 15 mg/kg. The rectal temperatures of the animals were measured 19 h after yeast injection. Thereafter, the animals received oral doses (50–200 mg/kg) of the extract, or indomethacin (5 mg/kg).The rectal temperatures were recorded again at intervals of 60, 90, and 120 min after extract administration.

Statistical analysis

Values are expressed as mean ± SEM. Statistical significance was determined using the Student's t.-test. Values with p < 0.05 were considered significant.

Results and Discussion

The extract of E. abyssinica. produced a dose-dependent and statistically significant (p < 0.05) inhibition of the migration of both total white blood cells as well as the polymorphonuclear leukocytes in reponse to carrageenan. A dose of 200 mg/kg of the extract produced a stronger neutrophil migration than indomethacin (5 mg/kg) (Table 1). These results further confirmed our earlier report on the ability of the plant extract to inhibit carrageenan-induced rat paw edema (Olajide & Alada, 2001). It also established the efficacy of the plant extract in preventing the events (that is, leukocyte migration) that tend to prolong or maintain the inflammatory response.

Table 1.. Effect of the methanol extract of E. abyssinica. leaves on leukocyte migration induced by carrageenan.

		Leukocyte migration^a. (×10^6 cells/ml)
Treatment	Dose (mg/kg)	Total	Neutrophils (PMNs)
Control	Saline	8.2 ± 0.4	3.2 ± 0.2
E. abyssinica.	50	6.2 ± 0.4^b.	2.7 ± 0.8^b.
E. abyssinica.	100	4.9 ± 0.2^b.	2.1 ± 0.1^b.
E. abyssinica.	200	3.7 ± 0.3^b.	2.0 ± 0.1^b.
Indomethacin	5	2.9 ± 0.4^b.	2.3 ± 0.2^b.

^a.Values are mean ± SEM of six independent observations.

^b.Values are statistically significant at p < 0.05, comparing each extract or drug treatment value with saline (control) values using the Student's t.-test.

Table 2 shows that the extract inhibited croton oil–induced ear edema in mice at a concentration of 20 mg/ear. This clearly indicates that E. abyssinica. has the potential of acting to inhibit topical inflammation when applied to the surface of the skin.

Table 2.. Effect of the methanol extract of E. abyssinica. leaves on croton oil-induced ear edema in mice.

Treatment	Concentration (mg/ear)	Edema (mg)^a.	Inhibition (%)
Control	—	13.8 ± 0.8	—
E. abyssinica.	5	13.2 ± 0.9	4.0
E. abyssinica.	10	13.3 ± 1.0	3.6
E. abyssinica.	20	8.7 ± 0.6^b.	40.0
Indomethacin	100 µg/ear	7.2 ± 0.7^b.	47.8

^a.Values are mean ± SEM of six independent observations.

^b.Values are statistically significant at p < 0.05, comparing each extract or drug treatment value with saline (control) values using the Student's t.-test.

In the experiment on the effect of the extract on acetic acid–induced writhing response in mice (Table 3), it was shown that only 100 and 200 mg/kg produced statistically significant (p < 0.05) effects. The number of writhing responses in mice given only saline was 56 ± 2.1, whereas it was 35.6 ± 1.9 and 21.0 ± 2.5 in the 100 and 200 mg/kg extract treated groups, respectively. The extract was ineffective in reducing the number of paw lickings in the early phase of the phenomenon induced by formalin (Table 4). However, there was a marked dose-related reduction in paw licking by the same mice in the late phase. The number of lickings in the phase by animals given only saline was 24.6 ± 2.3, whereas it was 14.2 ± 1.3, 11.8 ± 1.0, and 10.0 ± 0.6 in animals given 50, 100, and 200 mg/kg of the extract, respectively.

Table 3.. Effect of the methanol extract of E. abyssinica. on acetic acid–induced writhing in mice.

Treatment	Dose (mg/kg)	Number of writhings^a.	Inhibition (%)
Control	Saline	56 ± 2.1	—
E. abyssinica.	50	58.4 ± 2.6	−4.3
E. abyssinica.	100	35.6 ± 1.9^b.	36.4
E. abyssinica.	200	21.0 ± 2.5^b.	62.5
Indomethacin	5	10 ± 1.4^b.	82.1

^a.Values are mean ± SEM of six independent observations.

^b.Values are statistically significant at p < 0.05, comparing each extract or drug treatment value with saline (control) values using the Student's t.-test.

Table 4.. Effect of the methanol extract of E. abyssinica. leaves on formalin-induced paw licking in mice.

		Number of paw lickings^a.
Treatment	Dose (mg/kg)	Early phase	Late phase
Control	Saline	65 ± 4.7	24.6 ± 2.3
E. abyssinica.	50	56.4 ± 2.2	14.2 ± 1.3^b.
E. abyssinica.	100	54.0 ± 2.0	11.8 ± 1.0^b.
E. abyssinica.	200	56.0 ± 3.6	10.0 ± 0.6^b.
Indomethacin	5	17.8 ± 1.6^b.	9.2 ± 2.3^b.

^a.Values are mean ± SEM of six independent observations.

^b.Values are statistically significant at p < 0.05, comparing each extract or drug treatment value with saline (control) values using the Student's t.-test.

The observations in the two analgesic experiments show that E. abyssinica. is only effective in inflammatory pain and is ineffective in pains of neurogenic origins. The formalin test possesses two distinct phases. The earlier phase reflects a direct effect of formalin on nociceptors (noninflammatory or neurogenic pain), whereas the late phase reflects inflammatory pain (Elisabetsky et al., 1995). Antipyretic effect was not observed with the extract. All the mice treated with the extract had their rectal temperatures elevated considerably as much as those given only saline.

This study has demonstrated that the leaves of Entada abyssinica. possess both anti-inflammatory and peripheral analgesic effects. Follow-up studies would involve the isolation of bioactive compounds from the plant.