MATERIALS AND METHODS

Plant Material
The flowers of Newbouldia laevis for this study were collected from Kudingi Village-Samaru Zaria, in the month of August 2005. The studies were carried out in May 2007 and the plant specimen was identified by Mr. U.A. Gallah of the Herbarium Unit, Department of Biological Sciences, Ahmadu Bello University, Zaria-Nigeria, where a voucher specimen was deposited.

Extraction Procedure
The plant material was dried under shade for several days and then pulverized into fine powder. About 200 g of the powdered material was extracted exhaustively with 95% (v/v) ethanol in water using continuous soxhlet apparatus. The extract was concentrated under reduced pressure to yield a dark green mass that weighed 38.51 g (19.26% w/w). The crude ethanolic extract was then coded NLFE-Newbouldia leavis flower extract.

Animals
A total of fifty animals were used for these tests, comprising of twenty five adult male Swiss albino mice that weighed between 18-28 g and also twenty five male Wister rats which weighed between 150-250 g were obtained from the Animal house, Department of Pharmacology and Clinical Pharmacy, ABU Zaria-Nigeria. The animals were kept under well-ventilated conditions with 12 h light/dark cycle (6:00 am-6:00 pm) at room temperature, fed on Standard feeds (Excel feeds Plc. Kaduna, Nigeria) and allowed water ad libitum.

Drugs
Ketoprofen (Pfizer, USA), carrageenan, Sigma Chemical (Germany) and acetic acid, BDH reagent (Poole, UK); were obtained from a retail store at Samaru-Zaria, Nigeria. All drugs or reagents were freshly prepared to the desired concentration with distilled water or normal saline just before use.

Acetic Acid-Induced Writhing in Mice
This test was conducted employing the method described by Koster et al. (1959), Vongtau et al. (2000) and Usman et al. (2005). Swiss albino mice were divided into 5 groups of five mice each. Groups 1, 2 and 3 received 50, 100 and 200 mg kg^-1 body weight of the extract respectively. The fourth group served as control and was given normal saline equivalent to vehicle given with the extract; while the fifth group was given ketoprofen (10 mg kg^-1 body weight). The drug and extract were administered by intraperitoneal (i.p.) route. Thirty minutes later, all the groups were treated with acetic acid (0.06%, l mL per 100 g i.p.). Thereafter, mice were placed in an individual cage; the numbers of abdominal constrictions were counted five minutes after acetic acid injection for a period of 10 min. Percentage inhibition of writhing was obtained using the formula:

(Vongtau et al., 2000; Usman et al., 2005).

Carrageenan-Induced Paw Oedema in Rats
The test was conducted according to the method described by Winter et al. (1962). Wister rats were divided in to five groups each containing five rats. Groups 1, 2 and 3 received 50, 100 and 200 mg kg^-1 body weight of the extract respectively. The fourth group served as control and was given normal saline equivalent to vehicle given with the extract; while the fifth group was given ketoprofen (10 mg kg^-1 body weight). The drug and extract were administered by i.p. route. Thirty minutes later, 0.1 mL of freshly prepared carrageenan suspension (1% w/v in 0.9% normal saline) was injected into the sub planter region of the left hind paw of each rat. The paw diameter was measured with the aid of a vernier caliper at 1, 2, 3, 4, 5 h after injection of carrageenan.

Statistical Analysis
All values were expressed as mean±Standard Error of the Mean (SEM). The data of acetic acid and carrageenan tests were analysed statistically by one way analysis of variance (ANOVA) and Student’s t-test respectively. The differences between means were considered significant when p<0.05 and p<0.001.

RESULTS

Anti-Nociceptive Study
The extract at doses between 50 and 200 mg kg^-1 significantly decreased the number of acetic acid-induced writhes as shown in Table 1. The highest percentage inhibition of 83.74% was observed at extract dose of 200 mg kg^-1 while that of 50 mg kg^-1 showed 63.41%, indicating a dose-dependent activity pattern; the extract at the dose of 200 mg kg^-1 had similar activity profile to that of ketoprofen 10 mg kg^-1 exhibiting same percentage inhibition. This result showed that the extract (200 mg kg^-1) had exhibited a similar protection to that of the standard drug. All the values were significant (p<0.001) compared to the negative control (normal saline group).

Anti-Inflammatory Activity
The anti-inflammatory activity of flower extract of Newbouldia laevis against acute paw oedema (carrageenan-induced) in presented in Table 2 and 3 and the results showed that NLFE caused a slight inhibition of carrageenan-induced oedema over a period of 5 h. This effect appeared to be dose-dependent.

Table 1:	Effects of ethanolic extracts of Newbouldia laevis flower on acetic acid-induced writhing in mice
One way ANOVA; df = 4, 24; F = 42.558; n = 5; *: p<0.001

Table 2:	Effect of ethanolic extracts of Newbouldia laevis flower on carrageenan-induced oedema in rats
*: p<0.05; **: p>0.05 along each column

Table 3:	Percentage inhibition expressed by ethanolic extract of Newbouldia laevis flower and ketoprofen on carrageenan-induced oedema in rats

Maximum inhibitory effects of 55.0% was observed 5 h post treatment with the extract at a dose of 100 and 200 mg kg^-1, the drug (standard ketoprofen) indicated 80.3% as the maximum inhibitory effect 2 h post treatment. However, the mean percentage inhibition of these effects was found to be 33.14, 38.70 and 59.72 for 100, 200 and 10 mg kg^-1 extract and ketoprofen respectively. All the results were significant (p<0.001) compared to normal saline group (control).

DISCUSSION

Earlier reports on the phytochemical constituents of this extract (Usman et al., 2007a) revealed the presence of cardiac and steroidal glycosides, flavonoids, tannins among others while phytochemical constituents such as alkaloids, saponins was not detected. The earlier report on the acute toxicity studies by Usman et al. (2007a), revealed that the extract had an intraperitoneal LD₅₀ of 1264.9 mg kg^-1 in mice and thus, found the toxicity index of the extract in mice to be relatively moderately.

The method employed in this study also called abdominal constriction response, is very sensitive and able to detect anti-nociceptive effects of compound(s) at dose level that may be inactive in other methods like tail-flick test (Collier et al., 1968; Bentley et al., 1981; Usman et al., 2005, 2007b). The abdominal constrictions response is postulated to partly involve local peritoneal receptors (Bentley et al., 1983; Usman et al., 2007b). The extract NLFE significantly (p<0.001) reduced the number of abdominal constriction induced by acetic acid in mice. The effects observed were in a dose-dependent manner between 50-200 mg kg^-1; the higher dosage (200 mg kg^-1) exhibited similar activity as presented by ketoprofen 10 mg kg^-1. The activity was in line with the findings of Usman et al. (2007b) that as the concentration of these plant bioactive components increases, so also protection against abdominal writhes induced by acetic acid. It is therefore, pertinent to say that the extract contains active analgesic compound(s) when isolated in its pure form.

The carrageenan-induced oedema is commonly used as an experimental animal model for acute inflammation that has been believed to be biphasic, the first of which is mediated by the release of histamine and 5-HT and the release of kinin and prostaglandins in the later phase (Castro et al., 1968; Mazumder et al., 2003). In this study, the mean paw diameters of the rats were measured to determine the extent of protection by the tests samples. The results from this study indicated that the mean anti-inflammatory activity was found to be dose-dependent; significant differences (p<0.05, p<0.01 and p<0.001) between normal saline and the extract treated groups were observed at the early hours of treatment (1-2 h) and later at fifth hour post treatment; but no significant differences was noticed at the third and fourth hour (except 200 mg kg^-1 which was significantly different at p<0.05). There was significant differences observed throughout the tests between normal saline and the ketoprofen treated groups. The trend of the activities of the extract and that of ketoprofen was found to be similar at the earlier stages of the study; since the onset of protection were both observed at the second hours post treatment, while the anti-inflammatory activity of the extract behaved sinusoidally and reached peak at the fifth hour; that of the drug (ketoprofen) declined in a normal distribution pattern in later hours after treatment. Thus, the Mechanism of Actions (MOA) of the analgesic and anti-inflammatory effects expressed by the extract studied could be said to have similar properties and MAO with some groups of analgesic/anti-inflammatory agents (NSAIDS) including ketoprofen.

The co-existence of both anti-nociceptive and anti-inflammatory effects seen with this extract is well defined for various Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) particularly Salicylates and their derivatives (Musa et al., 2007). It is therefore, interesting to note that the extract behaves like NSAIDS, since it was able to potently exhibit anti-inflammatory effects at the later phase just as does prostaglandins It is hence, possible that both the analgesic and anti-inflammatory effects observed with this extract may be attributed to its flavonoids and/or tannins as reported earlier by Ahmadiani et al. (1998), Ahmadiani et al. (2000), Viana et al. (2003) and Usman et al. (2005), including some other phytochemicals. Flavonoids have also been reported to potently inhibit prostaglandins, a group of powerful pro-inflammatory signalling molecules (Manthey, 2000). Although, the exact chemical constituents responsible for these effects exhibited by this extract still remain speculative; the activities could be relevant to those earlier reported by similar secondary metabolites. Meanwhile, as far as we know, this is the first report on the analgesic and anti-inflammatory effects of extract from the part of this plant.

CONCLUSIONS

From the results obtained, it can be concluded that though relatively toxic, the plant extract possesses both anti-nociceptive and anti-inflammatory activity dose-dependently. These effects could therefore, be a point for the use of the plant traditionally as a remedy for rheumatic and other pain types. The authors wish to suggest that, further work be carried out on this plant extract with the view of isolating and characterizing the possible phytochemical agent(s) responsible for such activities.