Analgesic and anti-inflammatory effects of the methanol stem bark extract of Prosopis africana

Abstract

Prosopis africana (Guill. & Perr.) Taub. (Mimosoideae) is a shrub used for menstrual and general body pain in Nupe land in north central Nigeria. In this study, the methanol extract of the stem bark of Prosopis africana (at doses of 62.5, 125, and 250 mg/kg) was evaluated for analgesic and anti-inflammatory activities using acetic acid-induced writhing assay and carrageenan-induced inflammation in rats. The extract significantly (P <0.05) attenuated the acetic acid-induced writhing with the highest activity observed at the highest dose, 250 mg/kg (76.89%) comparable to that of piroxicam (83.16%) the standard agent used. In the carrageenan-induced inflammation assay, the extract showed significant anti-inflammatory activity (P <0.001) from the third hour. The preliminary phytochemical screening revealed the presence of flavonoids, saponins, carbohydrates, cardiac glycosides, tannins, and alkaloids. The oral median lethal dose was found to be 3807.9 mg/kg in mice and > 5000 mg/kg in rats. This study supports the folkloric claim of the use of Prosopis africana in the management of pain.

Keywords::

• Prosopis africana
• analgesic
• anti-inflammatory

Introduction

Prosopis africana (Guill. & Perr.) Taub. also known as African mesquite belongs to the family of Leguminosae, subfamily Mimosoideae. The common vernacular names of the plant include “kirya” (Hausa), “ayan” (Yoruba), “ubwo” (Igbo), and “sanchi” (Nupe). The plant, which is a quick-growing tree about 17 m tall is native to intertropical Africa, occurring from Senegal to Ethiopia throughout the Sudanese and Guinean ecozones, reaching the border of the Sahelian ecozone to the north. Prosopis africana is valuable for its thick wood. The red dye substance in the bark contains tannins and is used in many territories for tanning leather and cloth (Burkhill, 1995). The bark decoction is used locally in Senegal for opthalmias and toothache. The crushed bark is placed over skin infections and taken by draught for intestinal worms. The leaves are used for the treatment of migraine, vertigo, dysentery and rheumatism (Burkhill, 1995). Its seeds are used as a condiment in foods by various cultures. The roots are used for body vermin and bronchitis (Burkhill, 1995). The stem is used as a chewing stick for hardening of the teeth. The decoction of the stem bark is claimed to have pain relief properties in general body pain and menstrual pain by the Nupe people of Nigeria (Mann et al., 2003).

This study was carried out to evaluate the analgesic and anti-inflammatory properties of the methanol stem bark extract of Prosopis africana in a bid to establish scientific rationale for the folkloric use of this plant in painful conditions.

Materials and methods

Plant material

Prosopis africana was collected from Likoro village in the Kudan local government area of Kaduna state, Nigeria in September 2007. The plant was identified and authenticated by Mallam Musa and Umar Gallah at the herbarium section of the Department of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria, and a voucher specimen (6908) was preserved at the herbarium for future reference.

Preparation of the extract

The stem bark was cleaned, air dried at room temperature for seven days and size reduced to obtain a coarse powder using a pestle and mortar. The powdered material was then subjected to Soxhlet extraction over 24 h using methanol (100%). The extract was concentrated at a low temperature of 60°C using a water bath. This extraction procedure gave a yield of 31.22% w/w and was reconstituted into a fresh aqueous solution prior to each experiment.

Phytochemical screening

Phytochemical screening of Prosopis africana was carried out on the methanol stem bark extract using standard methods of analysis (Trease & Evans, 2002).

Test animals

Swiss albino mice (18-28 g) and Wistar rats (138-196 g) of either sex were used for the study. The animals were obtained from the animal house facility of the Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria. They were kept in standard animal cages at constant temperature (25° ± 1°C) and submitted to a 12 h light/dark cycle with a standard diet and water ad libitum. The animals were fasted 12 h prior to each experiment. All experimental procedures were approved by the ethical committee of the faculty.

Drugs and dosage

The drugs used in this study include piroxicam (Hovid 10mg/kg), 0.6% solution of acetic acid (Searle, Essex; 10 mL/kg), 1% solution of carrageenan (Sigma, St. Louis, MO, 0.1 mL/animal). Crude methanol stem bark extract of Prosopis africana (62.5, 125, and 250 mg/kg). Normal saline was used as control for each experiment. All test doses were administered orally.

Acute toxicity study

The median lethal dose (LD₅₀) was determined using the method previously described by Lorke (1983). In the first phase mice were divided into three groups with three mice in each group and were administered with the methanol stem bark extract at doses of 10, 100, and 1000 mg/kg body weight respectively via the oral route. The mice were observed for signs of toxicity and death for 24 h. In the second phase three groups of one mouse each were treated with more specific doses (5000, 2900, and 1600 mg/kg) of the extract respectively and observed for signs of toxicity and death in 24 h. The final LD₅₀ was calculated as the geometric mean of the lowest dose that caused death and the highest dose for which the animal survived. LD₅₀ was also determined in rats using the same procedure.

Acetic acid induced writhing

The method according to Koster et al. (1959) was employed for this test. Five groups of five mice each were pretreated with the extract (62.5, 125, 250 mg/kg), piroxicam 10 mg/kg and normal saline (10 mL/kg) respectively. One hour later each mouse was injected with 0.6% acetic acid at a dose of 10 mL/kg body weight. The number of writhing responses was recorded for each animal during a subsequent 10 min period after a 5 min latency period using a tally counter and the mean abdominal writhes for each group was obtained. The percentage inhibition was calculated using the formula

Carrageenan-induced paw edema

The test was conducted according to the method described by Winter et al. (1962). Five groups of five rats each were pretreated with the extract (62.5, 125, 250 mg/kg), piroxicam 10 mg/kg, and normal saline (1 mL/kg) respectively. One hour later, 0.1 mL of freshly prepared 1% carrageenan suspension was injected into the subplantar region of the left hind paw of each rat. The paw diameter was measured using a vernier caliper at 0, 1, 2, 3, 4, and 5 h after the injection of carrageenan.

Statistical analysis

The data are expressed as mean ± SEM. The results were analyzed by one-way ANOVA followed by Tukey’s test for multiple comparisons. P values less than 0.05 (P < 0.05) were considered significant.

Results

The oral median lethal dose (LD₅₀) of the methanol stem bark extract of Prosopis africana was found to be 3807.9 and >5000 mg/kg body weight in mice and rats, respectively. The extract significantly (p <0.001) attenuated the acetic acid-induced abdominal constrictions in mice. The highest percentage inhibition of abdominal constriction was obtained at 250 mg/kg (76.8%) and was comparable to that of piroxicam, the standard drug used (83.1%) (See Table 1).

Table 1.  Effect of methanol stem bark extract of P. africana on acetic acid-induced writhing in mice.

Treatments	Dose (mg/kg)	Mean abdominal writhes ± SEM	% Inhibition
Normal saline	-	19 ± 2.58	-
P. africana	62.5	12.2 ± 1.02^a	35.79
P. africana	125	12.4 ± 1.08^a	34.73
P. africana	250	4.4 ± 6.8^b	76.84
Piroxicam	10	3.2 ± 0.97^b	83.15

^ap<0.05; ^bp<0.001 compared to negative control (normal saline).

The methanol stem bark extract of Prosopis africana significantly (p <0.001) inhibited the progressive increase in paw edema produced by carrageeenan as seen in the control (normal saline treated) group. The anti-inflammatory effect of Prosopis africana at 125 mg/kg was comparable to that of piroxicam 10 mg/kg and at the 5^th h, the percentage anti-inflammatory effect of Prosopis africana (70.8%) was even higher than that of piroxicam (58.3%) (See Table 2).

Table 2.  Effect of methanol stem bark extract of P. africana on carrageenan-induced paw edema in rats.

Treatments	Dose(mg/kg)	Mean paw diameter (cm)
		1h	2h	3h	4h	5h
Normal saline	-	0.18 ± 0.01(0)	0.32 ± 0.04(0)	0.29 ± 0.02(0)	0.26 ± 0.02(0)	0.24 ± 0.02(0)
P. africana	62.5	0.13 ± 0.03(27.7)	0.21 ± 0.04^NS(34.4)	0.17 ± 0.03^b(41.4)	0.17 ± 0.02^b(34.6)	0.14 ± 0.02^b(41.7)
P. africana	125	0.13 ± 0.03(27.7)	0.18 ± 0.03^a(43.8)	0.14 ± 0.02^c(51.7)	0.14 ± 0.02^c(46.2)	0.07 ± 0.02^c(70.8)
P. africana	250	0.18 ± 0.02(0)	0.19 ± 0.02^a(40.6)	0.17 ± 0.02^b(41.4)	0.15 ± 0.01^c(42.3)	0.11 ± 0.02^c(54.2)
Piroxicam	10	0.11 ± 0.01(38.9)	0.12 ± 0.01^b(62.5)	0.10 ± 0.02^c(65.5)	0.11 ± 0.00^c(57.7)	0.09 ± 0.01^c(58.3)

Figures in parentheses represent percentage anti-inflammatory activity.

^ap <0.05; ^bp <0.01; ^cp <0.001 compared to negative control (normal saline).

NS, statistically not significant.

Discussion

Acetic acid-induced writhing (though widely used for the evaluation of peripheral analgesia) is a non-specific model as central analgesics also show a positive response to it (Vogel & Vogel, 1997; Trongsakul et al., 2003). This test is very sensitive and able to detect antinociceptive effects of compounds at dose levels that may appear inactive in other methods such as tail flick test (Collier et al., 1968). Local peritoneal receptors are postulated to be partly involved in the abdominal constriction response (Bentley et al., 1981) which has in turn been associated with increased peritoneal fluid levels of prostaglandins (PGE₂ and PGF2α) and lipoxygenase products following intraperitoneal injection of acetic acid (Deradt et al., 1980; Levini et al., 1984; Dhara et al., 2000). The analgesic activity of the extract may therefore be due to either its action on the peritoneal receptors, the inhibition of the production of arachidonic acid metabolites or the inhibition of synaptic transmission of painful messages to the central nervous system.

The carrageenan model which is used to detect anti-inflammatory activity in acute and sub-acute inflammation is said to be biphasic with the first phase (at the first h) mediated by the release of histamine and 5HT (Crunkhon & Meacock, 1971) and the latter phase (second to third h) due to the release of kinins and prostaglandins (Castro et al., 1968; Mazumder et al., 2003). From the results obtained from this study, the anti-inflammatory activity of the extract may be due to the inhibition of prostaglandins and kinins since its activity started from the second h. The carrageenan model can be used to indicate not only degree but also duration of anti-inflammatory action (Vogel & Vogel, 1997). The extract appears to have a long duration of anti-inflammatory action, evidenced by the fact that the anti-inflammatory activity of all test doses increased with time and also the extract at 125 mg/kg produced a higher anti-inflammatory activity than piroxicam, the standard drug used.

The coexistence of both analgesic and anti-inflammatory effect seen with this extract is well defined for various non-steroidal anti-inflammatory drugs (NSAIDs) since the cyclooxygenase enzyme which leads to the production of prostanoids is usually inhibited. Our results suggest that the extract has NSAID-like activity which correlates with the traditional use of the plant.

The preliminary phytochemical screening revealed the presence of flavonoids, tannins, glycosides, carbohydrates, saponins and alkaloids. Flavonoids, saponins and tannins have been shown to exert analgesic effects in the acetic acid-induced writhing test (Calixto et al., 2000). Saponins are reported to have anti-inflammatory effects supported by the reduction of carrageenan-induced lipid peroxidation and hydroxyl radical content in rat serum (Jongwon et al., 2005). Certain flavonoids possess potent inhibitory activity against a wide array of enzymes and prostaglandins (Midddleton, 1998; Manthey et al., 2001). Tannic acid and polyphenols have been shown to be potent inhibitors of nitric oxide synthetase activity and nitric oxide production which has been implicated in tissue damage and inflammation (Srivastava et al., 2000). Some tannins have been shown to inhibit the inflammatory events which occur at the onset of haemorrhagic cystitis (Viana et al., 2003).

The result of the investigation suggests that the methanol stem bark extract of Prosopis africana does possess significant analgesic and anti-inflammatory activities in laboratory animals and this supports the ethno-medical use of the plant in painful and inflammatory conditions.

Acknowledgements

The authors would like to thank Mrs. Rose Sangodare and the National Research Institute for Chemical Technology, Zaria, Nigeria for their assistance with the extraction of the plant.

Declaration of interest

There is no conflict of interest with regards to this study.