Anti-inflammatory Properties of Amaranthus spinosus Leaf Extract

Abstract

The methanol extract of Amaranthus spinosus L. leaves was evaluated for anti-inflammatory activities in different animal models. The effect of the plant extract was also studied on castor oil–induced diarrhea and gastric mucosal integrity. The extract (25–100 mg/kg) inhibited the carrageenan-induced rat paw edema and produced significant (p < 0.05) inhibition of acetic acid–induced increased vascular permeability. Inhibition of the cotton pellet granuloma was also inhibited by 100 mg/kg of the plant extract. Analgesic activity was exhibited with the significant and dose-related reduction in the number of writhings induced with acetic acid, as well reduction in paw licking induced by injection of formalin in mice. The extract (50 and 100 mg/kg) produced gastric erosion in rats, following repeated administration for 4 days, and with 25–100 mg/kg of the extract, there was a statistically significant (p < 0.05) reduction in castor oil–induced diarrhea in rats. These results demonstrate the anti-inflammatory properties of the leaf extract of A. spinosus. It is also suggested that the plant extract probably acts by the inhibition of prostaglandin biosynthesis.

Keywords:

• Amaranthus spinosus L. (Amaranthaceae)
• analgesic
• anti-inflammatory
• castor oil
• gastric mucosal erosion
• prostaglandin

Introduction

Amaranthus spinosus L. (Amaranthaceae) is a common weed found by roadsides, in waste places, fields, and cultivated beds. The leaves of the plant are used locally for the topical treatment of eczema, gastroenteritis, gall bladder inflammation, boils, and abscesses. It is also used in the treatment of snakebites, colic menorrhagia, and arthritis (Iwu, 1993). The plant is known to contain triterpenes, lipids, and flavonoids. Although the plant itself is not edible, it belongs to a genus whose members are widely cultivated in Nigeria as vegetables.

Earlier, an extract of the leaves of A. spinosus was reported to produce about 73% inhibition of prostaglandin biosynthesis in vitro (Ibewuike et al., 1997). There is a need to validate the local use of this plant in inflammatory conditions and to substantiate the in vitro prostaglandin biosynthesis inibitory activity of the plant, using in vivo models. In this study, therefore, the anti-inflammatory activity of the methanol extract of the leaves in animals is described. The effect of the extract on the gastric mucosa was also investigated.

Materials and Methods

Plant materials

The leaves of A. spinosus were collected from the campus of the Obafemi Awolowo University, Ile-Ife, in the month of November. The plant samples were authenticated in the Herbarium, Faculty of Pharmacy, Obafemi Awolowo University (Ile-Ife, Nigeria), where voucher specimens were deposited. The plant samples were thereafter shade-dried and reduced to a powdery form.

Weighed amount of the powdered sample was extracted exhaustively in methanol for 72 h. The resulting extract was then concentrated over a water bath until a solid extract sample was obtained (yield 10.2%). The extract was prepared in 2.5% Tween 80 for pharmacological studies.

Animals

Male Wistar rats (180–200 g) and male albino mice (20–22 g), obtained from the Animal House, Faculty of Pharmacy, Obafemi Awolowo University (Ile-Ife, Nigeria), were used. The animals were maintained under standard conditions and fed standard pellet diet (Guinea Feeds Ltd., Benin, Nigeria) and water ad libitum.

Carrageenan-induced rat paw edema

Edema was induced according to Winter et al. (1962). Carrageenan (0.1 ml of 1% suspension) was injected into the subplantar region of the right hind paw of each rat. The extract (25–100 mg/kg) was administrated orally to rats 1 h before carrageenan. Control rats received 10 ml/kg Tween 80, and indomethacin (5 mg/kg) was used as a reference drug. Increase in linear paw circumference was taken as a measure of edema volume (Hess & Milonig, 1972; Olajide et al., 1997, 2000).

Acetic acid–induced vascular permeability in mice

The method of Whittle (1964) was used. One hour after oral administration of the extract (25–100 mg/kg), mice were injected with 0.25 ml (i.p.) of 0.6% solution acetic acid. Indomethacin (5 mg/kg) served as the reference drug, whereas control animals received 10 ml/kg Tween 80. Immediately after administration, 10 ml/kg of 10% Evan's blue was injected i.v. into all the animals. Thirty minutes after Evan's blue injection, the mice were sacrificed, and the amount of dye that leaked into the peritoneal cavity was measured spectrophotometrically at 610 nm.

Cotton pellet granuloma in rats

The method of Ismail et al. (1997) previously described by Olajide et al. (1999) was used. A sterilized cotton pellet weighing 30 mg was introduced subcutaneously into rats. They were then treated orally with the extract (25–100 mg/kg) for 4 consecutive days. Control animals received 10 ml/kg Tween 80, and indomethacin (5 mg/kg) was administered to animals in the reference group. On the fifth day, the animals were killed and the pellets removed, dried overnight at 60°C, and weighed.

Gastric erosion assay

Subacute gastric damage was evaluated by repeated dosing of rats. For 4 consecutive days, 25, 50, and 100 mg/kg of the extract, 40 mg/kg indomethacin, or 10 ml/kg Tween 80 were given by gavage, in the morning, to animals fasted for 15 h. On day 5, all the animals were sacrificed, and their stomachs were cut open and examined macroscopically (Aparicio, 1979). The number of rats showing hyperemia and/or gastric ulcers was determined.

Castor oil–induced diarrhea in rats

The method described by Singh et al. (1997) was used, with slight modifications. Rats were fasted for 24 h and housed in individual cages lined with white blotting paper. The extract of A. spinosus (25–100 mg/kg) was orally administered to animals in different groups. Animals in the reference group received atropine (1 mg/kg, p.o.), whereas animals in the control group received10 ml/kg Tween 80. One hour later, castor oil (1 ml/100 g)was administered orally, and the animals were observed for 24 h for the number of wet feces produced.

Acetic acid–induced writhings in mice

The extract (25–100 mg/kg) was administered orally 1 h before writhing induction with 10 ml/kg (0.6%) acetic acid in mice. The control group received 10 ml/kg Tween 80, whereas mice in the reference group received indomethacin (5 mg/kg). Writhings occurring between 5 and 15 min after acetic acid were counted (Koster et al., 1959).

Formalin-induced paw lickings in mice

The method of Huskaar and Hole (1987) was used. Formalin (20 μl, 1%) was injected into the left hind paw of mice 1 h after extract (25–100 mg/kg) administration. Control animals received 10 ml/kg saline, and indomethacin (5 mg/kg) was administered orally to animals in the reference group. Mice were observed for time spent licking the injected paw (licking time), and this was recorded. Animals were observed for the first 5 min postformalin (early phase) or for 10 min starting at the 20th min postformalin (late phase).

Statistical analysis

Data are expressed as mean±SEM and analyzed using the Student's t-test. Values of p < 0.05 were considered significant.

Results

Carrageenan-induced rat paw edema

The extract of A. spinosus produced statistically significant (p < 0.05) inhibition of the edema induced by carrageenan at 50 and 100 mg/kg doses of the extract. At the third hour postcarrageenan, edema was inhibited by 25%, 37.5% and 53.3% by 25, 50, and 100 mg/kg of the extract, respectively (Table 1).

Table 1 Effect of A. spinosus methanol leaf extract on carrageenan-induced rat paw edema.

		Paw size^a (cm)
Treatment	Dose (mg/kg, p.o.)	Before carrageenan	3 h after carrageenan	Inhibition of edema (%)
Control	Tween 80	2.03 ± 0.05	3.23 ± 0.07	—
A. spinosus	25	2.02 ± 0.06	2.92 ± 0.06^b	25.0
A. spinosus	50	2.05 ± 0.06	2.80 ± 0.05^b	37.5
A. spinosus	100	1.97 ± 0.06	2.53 ± 0.08^b	53.3
Indomethacin	5	2.02 ± 0.03	2.43 ± 0.05^b	65.8

^aValues are expressed as mean±SEM for six independent observations (n = 6).

^bValues are statistically significant at p < 0.05, compared with Tween 80 treatment (Student's t-test).

Cotton pellet granuloma

Implantation of sterile cotton pellets under the skin of rats given the vehicle (10 ml /kg Tween 80) for 7 days resulted in an increase in the weight of the pellets from an average value of 20±1.5 mg to 42.0±3.0 mg. In animals treated with A. spinosus extract (25 and 50 mg/kg), the increase in weight of implanted pellets followed similar trends as the untreated animals. However, when the dose of the extract was increased to 100 mg/kg, there was a significant (p < 0.05) reduction in the postimplantation weight of the cotton pellets, when compared with rats that received vehicle over the same period of time (Table 2).

Table 2 Effect of A. spinosus methanol leaf extract on cotton pellet granuloma formation in rats.

		Weight of cotton pellets^a (mg)
Treatment	Dose (mg/kg, p.o.)	Before implantation	After implantation	Inhibition (%)
Control	Tween 80	20.0 ± 1.2	42.0 ± 3.0	—
A. spinosus	25	20.0 ± 1.6	42.7 ± 3.4	− 1.7
A. spinosus	50	20.0 ± 1.2	40.3 ± 3.1	4.1
A. spinosus	100	20.5 ± 1.2	29.2 ± 2.3^b	30.5
Indomethacin	5	20.5 ± 1.3	22.2 ± 1.7^b	47.1

^aValues are expressed as mean±SEM for six independent observations (n = 6).

^bValues are statistically significant at p < 0.05, compared with Tween 80 treatment (Student's t-test).

Acetic acid–induced vascular permeability

Intraperitoneal administration of acetic acid into mice pretreated with vehicle resulted in the leakage of 62.8±4.6 μg Evan's blue from the capillaries into the peritoneal cavity. However, pretreatment with the extract of A. spinosus (25–100 mg/kg) resulted in a significant (p < 0.05) and dose-related reduction in the amount of dye leakage, in comparison with the control animals (Table 3). Increase in vascular permeability was inhibited by 27.9%, 48.0%, and 57.5% with 25, 50, and 100 mg/kg of the extract, respectively. Indomethacin (5 mg/kg) produced the highest inhibition (68.5%).

Table 3 Effect of A. spinosus methanol leaf extract on acetic acid–induced vascular permeability in mice.

Treatment	Dose (mg/kg, p.o.)	Concentration of dye leaking out of capillaries^a (µg)	Inhibition of dye leakage
Control	Tween 80	62.8 ± 4.6	—
A. spinosus	25	45.3 ± 2.5^b	27.9
A. spinosus	50	37.2 ± 2.2^b	48.0
A. spinosus	100	26.7 ± 2.7^b	57.5
Indomethacin	5	19.8 ± 1.6^b	68.5

^aValues are expressed as mean±SEM for six independent observations (n = 6).

^bValues are statistically significant at p < 0.05, compared with Tween 80 treatment (Student's t-test).

Gastric erosion assay

In this experiment, the plant extract was repeatedly administered to fasted rats for 4 consecutive days, using indomethacin as a reference. Macroscopic examination of the stomachs on the fifth day revealed that in animals that received 50 and 100 mg/kg of the extract, as well as indomethacin (40 mg/kg), there were severe gastric erosions. Rats that were given Tween 80 as well as 25 mg/kg of the extract did not present with gastric ulcers (data not shown).

Castor oil–induced diarrhea

In Table 4, the number of feces produced by rats given castor oil (1 ml/100 g, p.o.) was significantly reduced with extract (25–100 mg/kg) pretreatment, when compared with rats that received Tween 80 prior to castor oil administration.

Table 4 Effect of A. spinosus methanol leaf extract on castor oil–induced diarrhea in mice.

Treatment	Dose (mg/kg, p.o.)	Number of wet feces^a
Control	Tween 80	23.2 ± 2.5
A. spinosus	25	16.7 ± 1.5^b
A. spinosus	50	10.3 ± 1.0^b
A. spinosus	100	9.5 ± 1.3^b
Atropine	1	7.5 ± 0.8^b

^aValues are expressed as mean±SEM for six independent observations (n = 6).

^bValues are statistically significant at p < 0.05, compared with Tween 80 treatment (Student's t-test).

Acetic acid–induced writhings

Amaranthus spinosus extract caused a statistically significant (p < 0.05) and dose-dependent reduction in the writhing response following injection with acetic acid. The highest dose of the extract (100 mg/kg) exhibited an inhibition of writhings (56.2%) comparable to 5 mg/kg indomethacin (58.4%) (Table 5).

Table 5 Effect of A. spinosus methanol leaf extract on acetic acid-induced writhings in mice.

Treatment	Dose (mg/kg, p.o.)	Number of writhings^a	Inhibition of writhings (%)
Control	Tween 80	23.3 ± 3.2	—
A. spinosus	25	15.3 ±1.7 ^b	34.3
A. spinosus	50	12.5 ± 1.3^b	46.5
A. spinosus	100	10.2 ± 0.7^b	56.2
Indomethacin	5	9.7 ± 1.0^b	58.4

^aValues are expressed as mean±SEM for six independent observations (n = 6).

^bValues are statistically significant at p < 0.05, compared with Tween 80 treatment (Student's t-test).

Formalin-induced paw lickings in mice

In Table 6, it is shown that pretreatment with the extract of A. spinosus (25–100 mg/kg) could not produce a significant reduction of licking time in the early phase. The licking time was, however, reduced in mice that received indomethacin. In the late phase, a dose-dependent and significant (p < 0.05) reduction in licking time was observed in mice treated with A. spinosus extract (25–100 mg/kg), as well as in mice treated with indomethacin (5 mg/kg).

Table 6 Effect of A. spinosus methanol leaf extract on formalin-induced paw lickings in mice.

		Licking time^a
Treatment	Dose (mg/kg, p.o.)	Early phase	Late phase
Control	Tween 80	38.3 ± 3.0	24.5 ± 2.6
A. spinosus	25	37.7 ± 4.2	18.3 ± 1.7^b
A. spinosus	50	35.0 ± 2.7	15.8 ± 1.8^b
A. spinosus	100	37.7 ± 2.2	12.7 ± 1.5^b
Indomethacin	5	25.2 ± 2.2^b	10.0 ± 1.1^b

^aValues are expressed as mean±SEM for six independent observations (n = 6).

^bValues are statistically significant at p < 0.05, compared with Tween 80 treatment (Student's t-test).

Discussion

In this study, the methanol extract of the leaves of A. spinosus was found to exhibit anti-inflammatory activity by inhibiting the edema induced by the injection of carrageenan into the hind paw of rats. This test is used in predicting the efficacies of anti-inflammatory agents that act by inhibiting the mediators of acute inflammation (Di Rosa et al., 1971). Earlier, Ibewuike et al. (1997) reported the in vitro prostaglandin synthesis inhibitory activity of an extract of A. spinosus. This report, coupled with our findings in this study, suggest that the plant could be exerting its anti-inflammatory effects in the same manner as the nonsteroidal anti-inflammatory drugs (NSAIDs). The NSAIDs have been known to exhibit anti-inflammatory actions by the inhibition of prostaglandin biosynthesis (Vane, 1971).

The extract of A. spinosus also produced a dose-dependent inhibition of the acetic acid–induced increased vascular permeability in mice. This observation substantiates the results of the carrageenan-induced edema in the paw of rats, as both models involve the exudation of fluid from within the blood vessels. The plant extract inhibited the cotton pellet granuloma at 100 mg/kg, which indicates that the extract is more effective in acute than in chronic, established inflammation.

The acetic acid–induced writhings test is a highly sensitive and useful test for analgesic drug development, and it is a model of visceral pain (Vyklicky, 1979). The extract of A. spinosus exhibited analgesic activity in mice, by the inhibition of the writhing response to a comparable degree as indomethacin, an established NSAID.

In spite of the usefulness of the acetic acid–induced writhing assay in drug development, it is not a selective pain test. It gives false positives with sedatives, muscle relaxants, and other pharmacological activities (Elisabetsky et al., 1995). The formalin test, however, is sensitive to NSAIDs. The test possesses two distinct phases, reflecting different types of pain. The earlier phase reflects a direct effect of formalin on nociceptors (neurogenic pain), whereas the late phase reflects inflammatory pain (Hunskaar & Hole, 1995). The extract of A. spinosus lacked activity in the early phase but produced significant (p < 0.05) and dose-dependent inhibition of the late phase. This observation further reflects the effect of the plant extract on inflammatory events.

The gastric ulcerative effect of A. spinosus reflects the possible prostaglandin synthesis inhibitory activity of the plant, which may be a major mechanism of its anti-inflammatory activity. The nonselective NSAIDs are known to inhibit prostaglandin synthesis at both inflammatory sites and the gastric mucosa due to the nonselective inhibition of both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. COX-1 is the constitutive enzyme that maintains the integrity of the gastric mucosa, and the COX-2 enzyme is the inducible form at inflammatory sites.

Interestingly, A. spinosus extract also reduced the incidence of diarrhea that was induced with castor oil in rats. The delay of castor oil diarrhea in rats has been postulated by Awouters et al. (1978) as a new way to evaluate the inhibitors of prostaglandin synthesis in vivo. Strub and Müller (1979) reported that there was a strong correlation by NSAIDs in the ability to reduce toxicity due to i.v. injection of arachidonic acid, the prevention of castor oil–induced diarrhea, and the enhancement of the gastric mucosal erosion formation. These workers suggested that these results pointed to inhibition of cyclooxygenase as a common mechanism of action.

The results of these in vivo experiments on Amaranthus spinosus substantiate the in vitro observation of Ibewuike et al. (1997). Taken together, the two studies have shown that the inhibition of prostaglandin synthesis is a major mechanism by which the plant exerts an anti-inflammatory effect. The present study also justifies the native use of A. spinosus in inflammatory conditions. It is not clear, however, the degree to which the plant extract affects the COX-1 and COX-2 enzymes. The focus of follow-up studies will be to investigate the effects of the plant extract and the biologically active compounds isolated from it on the gene expressions of pro-inflammatory factors such as COX-2, iNOS, and/or activation of NF κB. This is important in view of the emerging roles of these factors in other conditions such as cancer and Alzheimer disease.