Evaluation of aqueous extract of Felicia muricata leaves for anti-inflammatory, antinociceptive, and antipyretic activities

Abstract

Context: Felicia muricata Thunb. (Nees) (Asteraceae) leaves are used in folklore medicine of South Africa as an oral remedy for pain and inflammation. However, the efficacy of the plant part is yet to be validated with scientific experiments.

Objective: The current study is an effort to investigate the anti-inflammatory, antinociceptive, and antipyretic activities of aqueous extract of F. muricata leaves.

Materials and methods: The phytochemical screening of aqueous extract of Felicia muricata leaves and the efficacy of the extract at the doses of 50, 100, and 200 mg/kg body weight was investigated in experimental animals using several models of inflammation (paw edema induced by carrageenan and egg albumin), nociception (acetic acid-induced writhing, formalin-induced pain and tail immersion), and fever (brewer’s yeast-induced hyperthermia).

Results: The extract contained alkaloids, flavonoids, tannins, saponins, and phenolics. The extract dose-dependently reduced (P <0.05) the number of writhes and stretches induced by acetic acid, number of licks induced by formalin, paw volumes induced by carrageenan and egg albumin. The reaction time by the tail of the extract-treated animals to the hot water also increased. The extract also reduced hyperthermia induced by brewer’s yeast. The highest dose (200 mg/kg body weight of the extract) produced the best result in all cases.

Discussion and conclusion: This study revealed that the aqueous extract of Felicia muricata leaves possessed anti-inflammatory, antinociceptive and antipyretic activities. These findings have therefore supported the use of aqueous extract of Felicia muricata leaves in the traditional medicine of South Africa as an oral remedy for pains, inflammation, and fever.

Keywords::

• Asteraceae
• anti-nociception
• anti-inflammatory
• antipyretic
• brewer’s yeast
• Felicia muricata

Introduction

The use of plants in various parts of the world for both preventive and curative purposes is an age-old tradition and is increasing empirically. With this upsurge however, a thorough scientific investigation of these medicinal plants is imperative, based on the need to provide information on their efficacies and toxicity risk. One such plant used in the folklore medicine of South Africa as an oral remedy for pain, inflammation and fever without scientific evaluation of its efficacy is Felicia muricata.

Felicia muricata Thunb. (Nees) (Asteraceae) is a small drought resistant perennial aromatic herb growing up to 0.2 m high. In the Eastern Cape Province of South Africa, the rural dwellers use the plant in the management of headache, pain and inflammation (Hutchings & Van Staden, 1994). The antibacterial and antifungal activities of the methanol and acetone extracts as well as the essential oil from this plant have been studied (Ashafa et al., 2008a, 2008b). Toxicological studies on the aqueous extract of F. muricata leaves at the doses of 50, 100, and 200 mg/kg body weight in Wistar rats for 14 days revealed that the extract possessed selective toxicity on the hematological and serum lipids parameters as well as the liver and kidney functional end points (Ashafa et al., 2009). Several studies have also been carried out on the molecular mechanism of chronic inflammation as well as prevention and mitigation by botanicals in South Africa (Naidoo et al., 2006; Angeh et al., 2007), but to the best of our knowledge, the anti-inflammatory, antinociceptive and antipyretic activities of aqueous extract of Felicia muricata leaves have not been investigated in animals. There is the need, therefore, to provide scientific evidence for the folkloric claim of the use of the plant as an oral remedy for inflammation, pain and fever.

The present study was undertaken to validate the acclaimed anti-inflammatory, antinociceptive, and antipyretic activities of aqueous extract (the form in which the plant is prepared in traditional medicine of South Africa) of Felicia muricata leaves, using albino mice and rats as models.

Materials and methods

Plant material

The plant material, collected in March 2008 from a single population growing within the premises of the Alice campus of the University of Fort Hare, South Africa, was authenticated by Tony Dold of the Selmar Schonland Herbarium, Rhodes University, South Africa. A voucher specimen (AshafaMed.2007/1) was deposited at the Giffen Herbarium of University of Fort Hare.

Drugs and chemicals

Carrageenan, indomethacin, morphine sulfate, Tween-80 and brewer’s yeast were products of Sigma-Aldrich (Steinheim, Germany). All other chemicals used were of analytical grade and were products of Merck (Bellville, South Africa).

Animals

Male albino rats (Rattus norvegicus) of Wistar strain weighing between 228.53–243.57 g and Swiss albino mice weighing between 18.20–22.47 g used for this study were obtained from the Animal House of the Agricultural and Rural Development Research Institute, University of Fort Hare. The animals were housed in clean aluminum cages placed in well-ventilated house conditions (temperature: 23 ± 1°C; photoperiod: 12 h natural light and 12 h dark; humidity: 45–50%). They were also allowed free access to Balanced Trusty Chunks (Pioneer Foods, Huguenot, South Africa) and tap water free of contaminants. The study was carried out following approval from the Ethical Committee on the Use and Care of Animals of the University of Fort Hare, South Africa.

Preparation of extract

The leaves were oven-dried at 40°C for 48 h and pulverized using a Fritsh pulverisette 14^® Rotor-speed mill (Laborgeraetebau, Germany). Powdered plant material (100 g) was extracted in 500 mL of distilled water for 48 h on an orbital shaker (SO1 Stuart Scientific, Stone, UK). The extract was thereafter filtered through Whatman no. 1 filter paper (Maidstone, UK) and freeze-dried using a Savant Refrigerated Vapor Trap (RVT4104, California, USA) to yield 9.28 g. This was then reconstituted in distilled water to give the required doses of 50, 100, and 200 mg/kg body weight used in this experiment.

Phytochemical screening

The screening for some chemical constituents of the plant leaves was carried out as described by Sofowora (1993) for alkaloids, steroids, phlobatannins, phenolics, flavonoids, saponins, cardiac glycosides and tannins. Quantitative analysis was carried out as described for phenolics (Edeoga et al., 2005), flavonoids, alkaloids, saponins and tannins (El-Olemy et al., 1994).

Antinociceptive activity

Acetic acid-induced writhing test

The writhing test was conducted on the mice according to the procedure described by Gaertner et al. (1999). Briefly, five groups of six mice each were intraperitoneally administered with 0.6% (v/v) acetic acid at a dose of 10 mL/kg body weight. A known volume (0.5 mL) of distilled water (negative control) and same volume of aqueous extract of  Felicia muricata leaves containing 50, 100, and 200 mg/kg body weight were orally administered to each animal (0.5 mL/rat), 30 min prior to treatment with acetic acid. Each mouse in the positive control group was pre-treated subcutaneously with morphine sulfate (5 mg/kg) before the administration of acetic acid. The writhings (abdominal constriction and hind limb stretching) induced by the organic acid in all the groups were recorded for a 30-min duration after a latency period of 5 min. The percentage antinociceptive activity was calculated according to the following expression:

where X is the average number of stretchings of the control per group and Y is the average number of stretchings of the test per group.

Formalin-induced nociception

The procedure described by Adzu et al. (2003) was used for the formalin-induced nociception. Briefly, pain was induced by administering 0.05 mL of 2.5% (v/v) formalin (40% formaldehyde) in distilled water to the subplantar region of the right hind paw of each rat (0.05 mL/rat). Rats (six per group) were thereafter orally administered with 0.5 mL aqueous extract of F. muricata leaves containing 50, 100, and 200 mg/kg body weight for 30 min before the administration of formalin. Each animal in the positive and negative control groups was subcutaneously administered with 0.5 mL containing 5 mg/kg morphine sulfate and 0.5 mL distilled water respectively. The animals were thereafter individually placed in a transparent Plexiglas cage (25 cm × 15 cm × 15 cm) observation chamber. The amount of time spent on licking the injected paw was indicative of pain. The number of licks from 0 to 10 min (first phase) and 15 to 60 min (second phase) after the administration of formalin was recorded.

Tail immersion test

The tail immersion test was conducted as described by Aydin et al. (1999). Briefly, about 3 cm of the tails of rats pre-treated with the extract, reference drug and distilled water were immersed in a water bath (B-480, Buchi, Flawil, Switzerland) maintained at a temperature of 55° ± 0.5°C. Within a few minutes, the rats reacted by withdrawing the tail. This reaction time was recorded with a stop-watch. Each animal served as its own control and six readings were obtained for the control at 0- and 10-min interval. The average of the values was designated as the initial reaction time (pre-treatment) (T_b). The reference drug (morphine sulfate, 5 mg/kg, s.c.), distilled water (0.5 mL/animal, p.o.) and the aqueous extract of F. muricata leaves (50, 100, and 200 mg/kg body weight, p.o.) were administered to the rats in the various groups of six animals each. The reaction time (T_a) for the animals in the test groups was recorded at intervals of 0.5, 1, 2, 3, 4, and 6 h after a 30-min latency period. The cut-off time (time of no response) was put at 2 min. The response latency between the onset of immersion and the withdrawal of the tail was recorded. The percentage antinociceptive activity was calculated from the expression:

Where Ta is the reaction time for each of the animals in the test groups at intervals of 0.5, 1, 2, 4 and 6 h; Tb is the initial reaction time.

Anti-inflammatory activity

Carrageenan-induced edema test

The method described by Mohajer et al. (2005) was adopted with slight modification. Briefly, indomethacin (10 mg/kg body weight), the vehicle (0.9% (v/v) normal saline in 3% Tween-80 (2 mL/kg body weight) and the aqueous extract of F. muricata leaves (50, 100, and 200 mg/kg body weight) were administered 30 min before the sub-plantar injection of carrageenan to the six animals in each group. Acute inflammation was induced by sub-plantar administration of 0.05 mL of 1% carrageenan in normal saline that contained Tween-80 to the right hand paw of each rat. The hind paw thickness from the ventral to the dorsal surfaces was measured with a micrometer screw gauge (SMC-20326, Sterling Manufacturing, Ambala Cantt, India), immediately prior to carrageenan administration and then at 0.5, 1, 2, 4, 6, and 24 h after dosing with the reference drug, extract and the vehicle. The difference in the paw thickness before and after carrageenan treatment was computed as the degree of inflammation.

Egg albumin-induced rat paw edema

Fresh egg albumin was used to induce acute inflammation as previously described (Adzu et al., 2003). Thirty rats were grouped into five and pretreated as follows: group 1 received orally 10 mL/kg body weight of normal saline and served as the negative control, groups 2–4 were orally administered with 50, 100, and 200 mg/kg body weight of the aqueous extract of F. muricata leaves respectively, while group 5 (positive control) received orally 10 mg/kg body weight of indomethacin. Thirty minutes later, acute inflammation was induced by subplantar administration of 0.1 mL of undiluted fresh egg albumin to the right hand paw of the rats. The volume of the hind paw was measured by micrometer screw gauge before and at 20-min intervals for a total period of 120 min. Edema formation was assessed in terms of the difference in the zero time paw volume of the injected paw and its volume at the different time intervals after egg albumin injection. The percentage inhibition of edema was calculated using the expression:

where: a is the mean paw volume of treated rats after egg albumin injection, b is the mean paw volume of treated rats before egg albumin injection, c is the mean paw volume of control rats after egg albumin, d is the mean paw volume of control rats before egg albumin injection.

Antipyretic activity

The method described by Brune and Alpermann (1983) was adopted with slight modification for the antipyretic study. Rats used in this experiment were maintained in an animal house at a controlled temperature of 31.5°C. Pyrexia was induced in the animals (that had been deprived of feeds for 6 h but were adequately supplied with water ad libitum) by subcutaneous administration of 15% (w/v) of brewer’s yeast in 0.9% saline solution at a dose of 10 mg/kg body weight to near the groin of the animals. Following the injection, the site was massaged in order to spread the suspension uniformly beneath the skin. The rectal temperature of the rats was measured before and 18 h after the brewer’s yeast injection by inserting a clinical thermometer (Panamedic, Cheonan Choongnam, Korea), 3–4 cm into the rectum. Only animals that showed a rectal temperature > 36°C measured 18 h after the administration of brewer’s yeast were included in the experiment. The animals were orally administered with the aqueous extract of F. muricata leaves (50, 100, and 200 mg/kg body weight), distilled water (negative control) and indomethacin (10 mg/kg body weight) and thereafter allowed a latency period of 30 min before their rectal temperature was measured at 0.5–6 h post-dosing.

Statistical test

Data were presented as mean ± SEM. Statistical differences between control and treated groups were tested by one way Analysis of Variance and Duncan Multiple Range Test, complemented by Student’s t-test. Percentage data were arcsine transformed to account for wide variations in the values before analysis. Differences were considered significant at P <0.05.

Results

Phytochemical constituent

Phytochemical screening of aqueous extract of F. muricata leaves revealed the presence of alkaloids (0.2 ± 0%), flavonoids (0.23 ± 0.03%), tannins (0.45 ± 0.04%), saponins (0.94 ± 0.06%), and phenolics (0.04 ± 0.01%) whereas steroids, phlobatannins and cardiac glycosides were not detected.

Acetic acid-induced writhing

All the doses of the extract (50, 100, and 200 mg/kg body weight) significantly reduced (P <0.05) the number of writhes induced by acetic acid in the animals (Table 1). In contrast, compared with the distilled water control group, the computed percentage inhibition of antinociceptive activity by the extract increased significantly. The 100 mg/kg body weight of the extract produced inhibition of antinociceptive activity that compared favorably with morphine-treated positive control animals whereas the value at 200 mg/kg body weight of the extract was higher than the animals treated with the morphine.

Table 1.  Effect of aqueous extract of Felicia muricata leaves on acetic acid-induced writhing in mice (n = 6, X ± SEM).

Treatment	No. of writhings within 30 min	Inhibition (%)
Vehicle 0.5 mL	32.50 ± 2.06^a	0
Morphine 5 mg/kg	15.80± 2.11^b	51.38 ± 0.22
Extract 50 mg/kg bw	22.41 ± 1.72^c	31.05 ± 0.16
Extract 100 mg/kg bw	16.68 ± 2.51^b	48.68 ± 1.48
Extract 200 mg/kg bw	8.06 ± 0.33^d	75.20 ± 2.66

Test values carrying superscripts b, c, and d that are different from the control superscript, a, are significantly different (P <0.05) while those with same superscript are not significantly different (P >0.05).

Formalin-induced nociception

The extract at all the doses investigated in this study significantly decreased the number of licks induced by formalin between 0–10 min as well as between 15–60 min observation periods (Table 2). These decreases were more prominent during the second phase of 15–60 min. In addition, the effect was best in the animals treated with the highest dose of the extract (200 mg/kg body weight) during the two phases.

Table 2.  Effect of aqueous extract of Felicia muricata leaves on formalin-induced pain in male Wistar rats (n = 6, X ± SEM).

Treatment	Number of licks 0-10 min		Number of licks 15-60 min
	Score of pain	% Inhibition	Score of pain	% Inhibition
Vehicle 0.5 mL	19.71 ± 0.14^a	0	13.51 ± 0.13^a	0
Morphine 5 mg/kg bw	8.20 ± 0.42^b	58.40 ± 4.11	5.40 ± 0.06^b	60.03 ± 4.28
Extract 50 mg/kg bw	13.10 ± 0.16^c	33.54 ± 2.08	8.60 ± 0.08^c	36.34 ± 3.14
Extract 100 mg/kg bw	8.90 ± 0.26^b	54.85 ± 3.18	5.80 ± 0.09^b	57.07 ± 1.11
Extract 200 mg/kg bw	5.20 ± 0.10^d	73.62 ± 2.05	3.40 ± 0.07^d	74.83 ± 2.40

Test values carrying superscripts b, c, and d that are different from the control superscript, a, are significantly different (P <0.05) while those with same superscript are not significantly different (P >0.05).

Tail immersion model

The extract at all the doses investigated in this study significantly increased the reaction time of the animals to the hot water (Table 3). Whereas the reaction time produced by the highest dose of the extract (200 mg/kg body weight) compared well with that of the reference drug between 0–3 h, those recorded between 4–6 h were significantly higher than the morphine-treated animals.

Table 3.  Effect of aqueous extract of Felicia muricata leaves on pain induced by hot water (tail immersion test) in male Wistar rats (n = 6, X ± SEM).

Treatment	Latency period (h)
	0	0.5	1	2	3	4	6
Vehicle 0.5 mL	2.07 ± 0.01^a	2.09 ± 0.02^a	2.14 ± 0.03^a	2.11 ± 0.03^a	2.09 ± 0.05^a	2.11 ± 0.06^a	2.10 ± 0.04^a
Extract 50 mg/kg bw	2.06 ± 0.02^a	4.28 ± 0.07^b	4.01 ± 0.04^b	4.41 ± 0.03^b	3.96 ± 0.03^b	4.18 ± 0.04^b	4.19 ± 0.04^b
Extract 100 mg/kg bw	2.05 ± 0.02^a	3.29 ± 0.06^c	3.98 ± 0.03^b	4.22 ± 0.05^b	4.01 ± 0.04^b	4.65 ± 0.06^c	4.37 ± 0.04^c
Extract 200 mg/kg bw	2.06 ± 0.03^a	3.22 ± 0.05^c	4.36 ± 0.07^c	4.24 ± 0.04^b	5.31 ± 0.03^c	5.50 ± 0.07^d	4.36 ± 0.07^c
Morphine 5 mg/kg bw	2.02 ± 0.06^a	3.25 ± 0.02^c	4.40 ± 0.06^c	4.25 ± 0.04^b	5.33 ± 0.05^c	4.16 ± 0.04^b	4.14 ± 0.09^b

Test values carrying superscripts b, c, and d that are different from the control superscript, a, down the column for each latency period are significantly different (P <0.05) while those with same superscript are not significantly different (P >0.05). Values at 0 designated as Ta in the text is the initial reaction time (pre-treatment reaction time). Values at 0.5, 1, 2, 3, 4 and 6 h designated as Tb are the reaction times following the administration of drug and extract. Each test value is expressed in minutes.

Carrageenan-induced edema

There was a gradual increase in the carrageenan-induced edema paw volume of rats in the distilled water-treated group and this was sustained throughout the experimental period (Table 4). In contrast, all the doses of the extract significantly inhibited the carrageenan-induced edema paw volume. For example, whereas the 100 and 200 mg/kg body weight of the extract commenced the reduction of the paw edema volume by 30 min after treatment, the 50 mg/kg body weight did not manifest this effect until after 2 h of administration. Although, the extract and indomethacin significantly reduced the carrageenan-induced edema paw volumes in the animals, these paw volumes were still visible after 24 h (Table 4).

Table 4.  Effect of aqueous extract of Felicia muricata leaves on carrageenan-induced edema in male Wistar rats (n = 6, X ± SEM).

Treatment	Differences in right and left paw diameter (mm)
	0.5 h	1 h	2 h	4 h	6 h	24 h
Vehicle 0.5 mL	1.44 ± 0.03^a (0.0)	2.07 ± 0.01^a (0.0)	2.33 ± 0.07^a (0.0)	2.62 ± 0.02^a (0.0)	2.83 ± 0.07^a (0.0)	2.61 ± 0.06^a (0.0)
Extract 50 mg/kg bw	1.01 ± 0.05^b (29.86)	1.18 ± 0.03^b (43.00)	0.98 ± 0.03^b (57.93)	0.85 ± 0.08^b (69.47)	0.79 ± 0.03^b (72.08)	0.73 ± 0.06^b (72.03)
Extract 100 mg/kg bw	0.87 ± 0.03^c (39.58)	0.78 ± 0.03^c (62.23)	0.70 ± 0.09^c (69.96)	0.62 ± 0.06^c (76.34)	0.55 ± 0.07^c (80.57)	0.52 ± 0.01^c (80.08)
Extract 200 mg/kg bw	0.79 ± 0.01^d (45.14)	0.74 ± 0.02^c (64.25)	0.67 ± 0.06^c (71.24)	0.60 ± 0.03^c (77.10)	0.52 ± 0.01^c (81.63)	0.46 ± 0.01^c (82.38)
Indomethacin 10 mg/kg bw	0.77 ± 0.01^d (46.53)	0.75 ± 0.04^c (63.77)	0.67 ± 0.42^c (71.24)	0.61 ± 0.01^c (76.72)	0.57 ± 002^c (79.86)	0.54 ± 0.05^c (79.31)

Percentage inhibitions are indicated in brackets. Test values carrying superscripts b, c, and d that are different from the control superscript, a, down the column for each latency period are significantly different (P <0.05) while those with same superscript are not significantly different (P >0.05).

Egg albumin-induced rat paw edema

The egg albumin increased the paw edema in all the treatment groups up to 40 min post dosing (Table 5). This pattern of increase was reversed 60 min after the administration of indomethacin and the extract. However, the reduction in the egg albumin-induced paw edema by the extract compared well with the indomethacin-treated animals from 60–120 min post-dosing. The 200 mg/kg body weight of the extract produced the highest percentage inhibition on the egg albumin-induced paw edema (Table 5).

Table 5.  Effect of aqueous extract of Felicia muricata leaves on egg albumin-induced edema in Wistar rats (% inhibition) (n = 6, X ± SEM).

Treatment	Test period (min)
	20	40	60	80	120
Vehicle 0.5 mL	12.83 ± 0.07a	13.94 ± 0.04^a	14.74 ± 0.09^a	14.95 ± 0.05^a	15.3 ± 0.08^a
Extract 50 mg/kg bw	12.15 ± 0.04^a	3.97 ± 0.04^b	11.74 ± 0.09^c	9.6 ± 0.05^b	9.3 ± 0.08^b
Extract 100 mg/kg bw	12.89 ± 0.05^a	13.92 ± 0.03^a	11.37 ± 0.09^b	9.37 ± 0.04b	9.24 ± 0.07^b
Extract 200 mg/kg bw	11.27 ± 0.06^b	13.54 ± 0.07^a	11.44 ± 0.05^b	8.92 ± 0.09^c	8.05 ± 0.03^c
Indomethacin 10 mg/kg bw	11.39 ± 0.04^b	13.44 ± 0.03^a	11.58 ± 0.02^b	9.31 ± 0.02^b	9.32 ± 0.04^b

Test values carrying superscripts b, c, and d that are different from the control superscript, a, down the column for each latency period are significantly different (P <0.05) while those with same superscript are not significantly different (P >0.05).

Antipyretic activity

The body temperature induced by the brewer’s yeast of the distilled water-treated animals increased progressively throughout the experimental period (Table 6). The various doses of the extract reduced the elevated body temperature of the animals at different periods of the experiment. For example, whereas the 50 and 100 mg/kg body weight of the extract reduced the brewer’s yeast-provoked elevated body temperature of the animals after 4 and 2 h post dosing respectively, the 200 mg/kg body weight of the extract and the indomethacin commenced the reduction of the elevated body temperature of the animals after 1 h and 30 min post administration respectively.

Table 6.  Antipyretic activity of aqueous extract of Felicia muricata leaves in male Wistar rats. (n = 6, X ± SEM).

Treatment	Body temperature (°C)
	Initial	after 18 h	0.5 h	1 h	2 h	4 h
Vehicle 0.5 mL	35.33 ± 0.05^a	36.83 ± 0.03^b	37.30 ± 0.04^c	37.73 ± 0.07^c	38.80 ± 0.09^d	38.82 ± 0.04^d
Extract 50 mg/kg bw	35.88 ± 0.06^a	36.40 ± 0.05^b	37.25 ± 0.02^c	37.65 ± 0.06^c	38.73 ± 0.05^d	35.50 ± 0.01^a
Extract 100 mg/kg bw	35.60 ± 0.03^a	36.20 ± 0.05^b	37.40 ± 0.05^c	37.95 ± 0.03^c	35.20 ± 0.05^a	35.60 ± 0.05^a
Extract 200 mg/kg bw	35.57 ± 0.08^a	36.70 ± 0.03^b	37.20 ± 0.03^c	35.55 ± 0.03^a	35.78 ± 0.05^a	35.63 ± 0.08^a
Indomethacin 10 mg/kg bw	35.53 ± 0.08^a	36.88 ± 0.06^b	35.22 ± 0.06^a	35.53 ± 0.09^a	35.63 ± 0.07^a	35.58 ± 0.05^a

Test values carrying superscripts b, c, and d that are different from the control superscript, a, down the column for each latency period are significantly different (P <0.05) while those with same superscript are not significantly different (P >0.05).

Discussion

Plant-based in vivo research has made significant and rewarding progress in many important areas such as male sexual dysfunction (Yakubu & Afolayan, 2009), and is still contributing to research on pain, inflammation and fever. Phytochemical screening has revealed many bioactive agents of plant extracts (Yakubu et al., 2005). For example, the anti-inflammatory property of flavonoids has been attributed to its influence on the production of prostaglandins via selective inhibition of cyclooxygenase type 2 (COX-2) as well as suppressing 5S-hydroxyeicosatetraenoic acid (Soojin et al., 2004). This activity leads to decreased production of prostaglandin. In addition, several studies have attributed the anti-inflammatory, antinociceptive and antipyretic activities of many plants to sterols, triterpenes, tannins, alkaloids, and/or flavonoids (Ghannadi et al., 2005; Silva et al., 2005; Chang et al., 2007).

Although acetic acid-induced writhing is a non-specific model, it is still widely used for antinociceptive screening. Acetic acid produces writhings and abdominal constrictions indirectly by stimulating the release of endogenous mediators such as prostaglandin (PGE₂ and PGE₂α) into the peritoneal fluid. This in turn enhances the sensitivity of nociceptive neurons sensitive to NSAID (Adzu et al., 2003). Thus, the dose-dependent inhibition of abdominal constrictions by the aqueous extract of F. muricata leaf indicates antinociceptive potential of the plant. The extract might have interfered with the mechanism of energy transduction in the primary efferent nociceptor (Sulaiman et al., 2008), or suppressed the synthesis of prostaglandin in the bodies of the animals.

The formalin-induced pain model is a more sensitive test for various classes of antinociceptive agents. It has been shown that the mechanism of formalin-induced pain in the early phase may involve sensorial C-fibers (Heapy et al., 1987), whereas a combined process generated by peripheral inflammatory tissue and functional changes in the dorsal horn has been associated with the late phase (Dalal et al., 1999). In addition, centrally acting drugs inhibit both phases (aphasic/neurogenic and tonic/inflammatory) equally, while peripherally acting drugs inhibit only the tonic phase (Shibata et al., 1989; Adzu et al., 2003). Although the extract inhibited both phases, it is not sufficient to conclude that the extract is central acting, since both phases were not inhibited equally. Therefore it is possible that central and peripheral mechanisms are involved in the antinociceptive activity of the plant. The higher percentage inhibition of pain produced during the second (tonic) phase suggested that the peripheral mechanism is more involved than the centrally acting process. This might also explain the anti-inflammatory activity of the extract, since inflammation is a peripheral process (Ahmadiani et al., 2000; Adzu et al., 2003). This agreed with the findings of Sulaiman et al. (2008) on aqueous extract of Ficus deltoidea Jack leaves.

The tail immersion model is used to determine acute pain. Centrally acting antinociceptive agents elevate pain threshold of animals towards heat and pressure (Adeyemi et al., 2004). Therefore, the elevated pain threshold of the animals by the aqueous extract of F. muricata leaves suggests that the extract might be a centrally acting analgesic. This agrees with the findings of Xie et al. (2008) on the anti-inflammatory and antinociceptive activities of two limonoids from the fruits of Melia toosendan Siebold and Zucc.

Carrageenan-induced rat paw edema is a suitable model for assessing the anti-edematous effect of natural products and it is believed to be biphasic (Adedapo et al., 2008). The first phase (1 h) involves the release of serotonin and histamine while the second phase (over 1 h) is mediated by prostaglandin, and the continuity between the two phases is provided by kinins (Perianayagam et al., 2006). This model is also a significant predictive test for anti-inflammatory agents acting via mediators of acute inflammation (Owoyele et al., 2005; Moody et al., 2006). In this study, the anti-edematogenic effects of the extract of F. muricata leaves were indications of anti-inflammatory potentials of the plant. The anti-inflammatory activity of the extract was supported earlier in this study by the formalin-induced pain model. This suggests that the extract could be used in managing acute inflammatory disorders as is done in folklore medicine of South Africa.

The use of egg albumin as is done phlogistic and screening agents for anti-inflammatory activity has been reported (Adzu et al., 2003). This test model was investigated in this study to complement the carrageenan-induced paw edema volume in rats. The ability of the aqueous extract of F. muricata leaves to suppress the exudative inflammation induced by the egg albumin suggests anti-inflammatory activity for the extract. Although the levels of pro-inflammatory mediators such as histamine, bradykinin and prostaglandin were not determined in this study, the anti-inflammatory activity of this extract may still be attributed to inhibitory effects on these pro-inflammatory mediators.

Elevation of body temperature (fever) may be the consequence of infection, one of the sequelae of tissue damage, inflammation, graft rejection, tumor necrosis factor-α (TNF-α) and prostaglandins (Kluger, 1991). Antipyretics have been shown to suppress fever either by inhibiting prostaglandin synthetase through the suppression of COX-2 (which results in the blockade of the synthesis of prostaglandin in the brain) or suppressing the rise of interleukin-1α subsequent to interferon production (Luo et al., 2005). Although this study revealed that different doses of the extract exhibited varying degree of antipyretic effect on the animals, the ability of the extract to lower brewer’s yeast-provoked hyperthermia suggests antipyretic potential of the extract. Alkaloids such as boldine have been reported to inhibit the synthesis of prostaglandin E₂ (Backhouse et al., 1994) which eventually reduced elevated body temperature in animals. Similarly, flavonoids such as biacalin have equally been reported to exhibit antipyretic effect by suppressing TNF-α (Chang et al., 2007). Therefore, the antipyretic effect of aqueous extract of Felicia muricata leaves in this study may be due to the presence of bioactive principles such as flavonoids and alkaloids present in the plant extract.

In conclusion, this study has demonstrated that the aqueous extract of Felicia muricata leaves exhibited anti-inflammatory, antinociceptive and antipyretic activities in the animal models. This however, may be due to the ability of the extract to suppress the release of endogenous mediators. The plant extract may also be useful as a centrally and a peripherally acting antinociceptive agent. This study thus supports the folkloric claim of aqueous extract of Felicia muricata leaves in the management of inflammation, pain and fever in South Africa.

Acknowledgements

The authors are grateful to the National Research Foundation of South Africa and the Govan Mbeki Research and Development Centre of the University of Fort Hare for their support.

Declaration of interest

The authors are also grateful to the University of Ilorin, Nigeria, for supporting the Postdoctoral Fellowship Programme of M. T. Yakubu at the University of Fort Hare, Alice, Eastern Cape, South Africa.