Analgesic and anti-inflammatory effects of the ethanol extract of Elytraria marginata Vahl ( whole plant ) in Wistar rats

The analgesic and anti-inflammatory effects of the ethanol extract of Elytraria marginata were studied using hot plate, thermal-induced pain and carrageenan-induced acute inflammation models in adult Wister rats. The preliminary phytochemical constituents of the extract were also ascertained. Inflammation was induced by injecting 0.1 ml of 1% carrageenan into the sub-planter surface of the right hind paw of the rats. Ethanol extract of E. marginata with doses of 50, 100 and 150 mg/kg and diclofenac 10 mg/kg were administered orally to separate groups of rats. Control group received 10 ml/kg of distilled water. The results showed that the extract (50, 100 and 150 mg/kg) significantly (p<0.05) reduced the carrageenan-induced rat paw oedema in a dose-dependent manner. The oedema reductions at a dose of 150 mg/kg at the 4th h were comparable to that obtained for diclofenac, the standard anti-inflammatory drug at the 3rd hour. The extract also showed a very good analgesic response against hot plate-induced analgesia (thermal stimuli) in the rats. Administration of the extract doses (50, 100 and 150 mg/kg) and ibuprofen 100 mg/kg produced a significant (p<0.05) reduction in the pain induced by the hot plate (thermal stimuli) in all the experimental groups when compared with the control. Preliminary phytochemical analysis showed the presence of alkaloids, flavonoids, tannins and saponins. These findings indicate that the ethanol extract of E. marginata has both analgesic and antiinflammatory effects and could be beneficial in alleviating painful inflammatory conditions.


INTRODUCTION
Inflammation is a pathophysiological reaction of living organisms to injuries that leads to the local accumulation of plasma fluids and blood cells.Though a defense mechanism, the complex events and mediators involved in the inflammatory reaction can induce, maintain, or intensify numerous diseases (Carey et al., 2009).
In recent times, attention to medicinal plants has increased globally, and a number of evidences have shown the immense potential of medicinal plants traditionally used in various parts of the world (Anosike et al., 2009).Medicinal plants are considered to be an important source of new chemical substances with potential therapeutic effects (Goji et al., 2010).However, studies on inflammatory diseases are gaining much responsiveness, and the side effects of the currently available anti-inflammatory drugs pose a major problem during their clinical use (Kayaalp, 1998).Hence, the development of newer and more powerful antiinflammatory drugs with less significant side effects is imperative.
The study of plants which are used traditionally in treating inflammatory conditions should be useful in the search for new drugs with anti-inflammatory and analgesic potentials.In Nigeria, many plant products are employed for their analgesic and anti-inflammatory effects and their efficiency is traditionally acclaimed.One of such plant is Elytraria.marginataVahl.(Acanthaceae).
E. marginata is locally known as "ewe eso" in Yoruba in Nigeria.It is an annual or short-lived perennial herb, the stem is up to 9 inches high, with small white or bluish flowers and it is widely found in Tropical Africa.The plant has been reported for its use in the treatment of measles in traditional practices in Nigeria (Sonibare et al., 2009), and has been reported as one of the plants used locally in the treatment of sterility in women in Ivory Coast (Assi, 1980).Djah and Nueba (2011) also reported that E. marginata is used traditionally by Anyi-Ndenye women during pregnancy to ensure good development of pregnancy and to facilitate labour.Elytrariaacaulis, another species of Elytraria has been reported to be an anti-hyperglycemic agent on the alloxan-induced diabetes in rats (Kumudhavalli and Jayakar, 2011).
Though the plant has been used in traditional medicine in treating inflammatory condition, there is no scientific evidence for such activities available in literature.The objective of the present study is therefore to investigate the analgesic and anti-inflammatory effects of the ethanol extract of E. marginata on carageenan-induced oedema and hot plate-induced analgesia in experimental models in order to validate its traditional use and determine the probable phytochemical constituents present in the ethanol extract of E. marginata as the basis for its pharmacological actions.

Plant collection and identification
The fresh plant of E. Marginata was purchased from Ago-Iwoye local market in August, 2012 and was identified by Mr. Shosanya, O.S. of the Forestry Herbarium at the Forestry Research Institute of Nigeria (FRIN), Ibadan, Oyo State, Nigeria.The voucher number allocated to the herbarium specimen is FHI 109760.

Experimental animals
A total number of 30 adult Wistar rats of both sexes were purchased from the Department of Physiology, College of Veterinary Medicine of the Federal University of Agriculture, Abeokuta, Ogun State, Nigeria.The animals weighed between 150 to 200 g.The animals were put under a well-ventilated condition and were housed in a cage of three animals each.They were allowed to acclimatize with the new environment before theexperiment commenced, and were adequately fed with pelletized feed and water ad libitum.

Preparation of the extract
The dried plant of E. marginata was ground with an electric blender.The ground plant, 0.45 kg was macerated in 3 L ethanol for a period of one week.The resulting solution was filtered and the filtrate was concentrated to dryness using a water bath at 50°C.After determination of percentage yield, the extract was stored in the refrigerator (4°C) until needed for analysis.

Test for alkaloid
To 0.2 g of the crude extract of E. marginata, 10 ml of 10%, HCl was added and placed on a water bath for 5 mins.The extract was then filtered and allowed to cool.The pH was then adjusted to about 6 to 7 by adding 10% ammonia and using litmus paper.Then 5 ml of the filtrate was taken into separate test tubes and small quantity of Wagner's, Mayer's and Dragendorff's reagents were added and observed.The presence of turbidity or precipitation indicates the presence of alkaloid (Sofowora, 1993).

Test for anthraquinone glycoside
To 0.2 g of the crude extract of E. marginata, 2 ml of 10% HCl was added and placed on a water bath for 5 mins and filtered while still hot, then allowed to cool.The filtrate was partitioned with equal volume of chloroform and shaken gently.The chloroform layer (lower layer) was transferred to a clean test tube and equal volume of the ammonia solution was added and then shaken gently.The presence of delicate rose-pink layer on the test solution indicated the presence of anthraquinones (Sofowora, 1993).

Test for cardiac glycoside
To 0.2 g of the crude extract was added water and 2 to 3 drops of lead acetate solution then was shaken gently and filtered.To the filtrate, 2 ml of chloroform was added and then 1 ml concentrated H 2 SO 4 was carefully added to form a lower layer.A reddish brown colour at interface was observed for cardiac glycoside (Sofowora, 1993).

Test for flavonoid
To 0.2 g of the crude extract of E. marginata, 10 ml of ethanol was added and 3 drops of Ferric chloride (FeCl 3 ) was added.A dark green colour indicated the presence of flavonoid (Ajaiyeoba, 2002).

Test for saponin
Crude extract of E. marginata (0.2 g) was placed in a test tube containing 10 ml of distilled water, and then boiled for f5 mins and then filtered.The filtrate was shaken vigorously and observed.The presence of froths indicated the presence of saponin (Sofowora, 1993).

Test for tannin
Crude extract of E. marginata (0.2 g) was decocted with 20 ml of distilled water by boiling for 10 mins and filtered while hot and allowed to cool.Ferric chloride reagent (0.1%) was added to the filtrate.A blue-black, green or blue green indicated the presence of tannins (Sofowora, 1993).

Test for cardenolides
Crude extract of E. marginata (0.2 g) was added 2 ml of glacial acetic acid containing a drop of FeCl 3 solution.Then 1 ml of concentrated H 2 SO 4 was gently added to form under layer.A brown ring indicated the presence of cardenolides (Sofowora, 1993).

Anti-inflammatory response
The carrageenan-induced acute inflammation model was employed (Winter et al., 1962).A total number of fifteen rats were fasted for 6 h and divided into 5 groups of 3 rats per group.Deprivation of water was to ensure uniform hydration and to minimize variability in oedematous response (Winter et al., 1963).Various doses of the extract (50, 100 and 150 mg/kg) were administered orally to groups 1 to 3, respectively.Group 4 received oral administration of distilled water (10 ml/kg) as negative control while group 5 received oral administration of diclofenac sodium (10 mg/kg) as positive control.After 1 h, 0.1 ml of freshly prepared 1% carrageenan suspended in distilled water was injected into the subcutaneous region of the right hind paw of the rats.The measurement of the paw was taken before (initial paw volume) and after the injection of the carrageenan at 0.5, 1, 2, 3, 4 and 5 h using a Vernier caliper.The mean paw circumference and percentage inhibition of oedema was determined using the methods of Okpo et al. (2001) and Akah (2009).The percentage inhibition was calculated using the modified formula used by Ahmad et al. (1992) below:

Percentage inhibition = (A-B)/A × 100
Where A is mean paw size of the control group and B is the mean paw size of the treated group.

Analgesic response (thermal stimuli)
Hot plate-induced analgesia was employed according to Ilodigwe and Akah (2009).Fifteen rats were fasted for 6 h and divided into 5 groups of 3 rats per group.The extract (50, 100 and 150 mg/kg) was administered orally to groups 1 to 3, respectively.Group 4 received oral administration of distilled water (10 ml/kg) as negative control while group 5 received oral administration of Ibuprofen (100 mg/kg) as positive control.After 1 h, each rat was gently placed on the hot plate maintained at 55±0.5°C and the time required by the rat to lick the paw or jump was taken as the response.The cut-off time or latency response was 15 seconds to avoid tissue damage.The percentage inhibition was calculated using the modified formula below:

Percentage inhibition = (A-B)/A x 100
Where A is mean increase in latency of the treated group and B is the mean increase in latency of the control group.

Statistical analysis
The results of the experiment were expressed as Mean ± Standard error mean (SEM).Numerical data were analysed for homogeneity of variance using Bartlett's test.The data were then analysed using one-way ANOVA to determine whether results in a particular group is significantly different from those in the corresponding control groups.The analysis of variance was followed by Tukey post hoc for intergroup comparisons.P<0.05 were considered as significant.

RESULTS
The preliminary phytochemical screening of the ethanol extract of E. marginata (Table 1) showed the presence of alkaloids, cardiac glycoside, flavonoids, saponins and tannins.Other constituents like anthraquinones and cardenolides were absent.

Carrageenan-induced oedema
The effect of E. marginata ethanol extract on carrageenan-induced oedema is shown in Table 2.The extract produced a dose-dependent inhibition of oedema induced by carrageenan.The percentage inhibition produced by the extract EM (150 mg/kg) was comparable to that produced by diclofenac sodium, 10 mg/kg (standard drug) at the 3rd h post-carrageenan injection.Highest percentage of the inhibitory effect of the extract (39.56%) was observed at a dose of 150 mg/kg body weight at the 5th h.This inhibitory effect was comparableto that exhibited by diclofenac sodium (40.91%) at 10 mg/kg body weight at the 4th h.

Effect on hot plate analgesia
A significant (p<0.05) and dose-dependent elevation of the treatment reaction time to thermal pain was evident in the extract-treated animals.The effect of the extract (150 mg/kg) was comparable to that produced by 100 mg/kg of ibuprofen (standard drug) as displayed in Table 3. Highest percentage of the inhibitory effect of the extract (72.46%) was observed at a dose of 150 mg/kg.The inhibitory effect was greater compared to that of Ibuprofen at a dose of 100 mg/kg (67.15%).

DISCUSSION AND CONCLUSION
The present study showed some of the pharmacological basis for the ethnomedicinal use of E. marginata in the treatment of inflammation.Carrageenaninduced inflammation is one of the most employed models reported for screening of clinically effective antiinflammatory agents.Oedema formation due to carrageenan in rat is a biphasic event.The initial phase of oedema is attributed to the release of histamine and serotonin, and the second phase of oedema is due to the release of prostaglandins, protease and lysosomal enzymes.However, it has been reported that the second phase is sensitive to the most clinically effective antiinflammatory agents (Thakare et al., 2009).The ethanol extract of E. marginata showed a good anti-inflammatory activity against acute inflammation induced by carrageenan in a dose-related manner at 3rd and 5th h.Oedema results from the action of inflammatory mediators such as histamine, serotonin and bradykinin at the site of a local inflammatory insult (Harriot et al., 2004).The early phase of oedema, beginning from 1 h after the administration of the irritant (carrageenan), is due to the release of histamine and serotonin, while the later phase, occurring from 3 to 5 h after the administration of the irritant (carrageenan) is induced by bradykinin, protease, prostaglandin and lysosome (Wallace, 2002;Harriot et al., 2004).The reduction in oedema exhibited by ethanol extract of the study plant showed that it contains active constituents which block the release of histamine and serotonin from mast cells and inhibit the activity of other inflammatory mediators.
From this study, E. marginata ethanol extract demonstrated significant (*p<0.05)and dose dependent anti-inflammatory activity against carrageenan-induced paw oedema.The effect of the extracts was most pronounced, 3 h after the induction of oedema, an action which was similar to that of diclofenac sodium, showing its usefulness in the management of acute inflammation.E. marginata (150 mg/kg) caused 39.56% inhibition of carrageenan-induced paw oedema compared to that of the standard drug (diclofenac sodium, 10 mg/kg) which was 40.91% at the 4th h.Thermal painful stimuli are selective for the evaluation of centrally, but not peripherally acting analgesic drugs (Chau, 1989).E. marginata (150 mg/kg) produced 72.46% pain inhibition compared to that of the standard drug (Ibuprofen, 100 mg/kg) which produced 67.15% pain inhibition.These results suggest that the extract possesses non-steroidal anti-inflammatory drugs-like analgesic activities, mediated through both the peripheral and central mechanisms.However, the result of the thermal stimuli (hot plate) experiment showed that the extract is more effective in alleviating central pain.Since pain is an integral part of inflammation, the analgesic activity shall certainly be a beneficial factor during inflammatory condition.The phytochemical screening of the plant showed that it contains alkaloids, flavonoids, saponins, tannins and phenols.To the best this study knowledge and within the scope of available literature, this is the first report of phytochemical screening on E. marginata.In a study on related species, Elytrariaacaulis, twopyrazole alkaloids were isolated and characterized to be with asomnine and 4'-hydroxywithasomnine (Ravikanth et al., 2001).In another study, asomnine was shown to have inhibitleukotriene metabolis mLTB4, and well had double inhibitory effect on COX-1 and COX-2 enzymes (Wube et al., 2008).Meanwhile, naturally occurring alkaloids, flavonoids and saponins have been found to elicit analgesic and anti-inflammatory properties (Fernanda et al., 2002;Anaga and Onehi, 2010).According to Manthey (2000), prostaglandins, a group of powerful pro-

Table 1 .
Result of phytochemical screening of the E. marginata ethanol extract.