Anti-inflammatory evaluation of methanol extract and aqueous fraction of the leaves of Anthocleista djalonensis A. Chev (Gentianaceae).
Abstract
The effects of the methanol extract (crude) and aqueous fraction of Anthocleista djalonensis on acute inflammation were examined. Oral administration of the methanol extract showed acute anti-inflammatory activity against Carrageenan induced paw edema in rats although not too significantly different (P>0.05) from the control at 250 mg/kg and 500 mg/kg. The aqueous fraction of the methanol extract significantly inhibited (P < 0.05) carrageenan induced paw edema in rats at 500 mg/kg. Significant activity against Dextran induced paw edema in rats was exhibited by both methanol extract (P< 0.01) and aqueous fractions (P< 0.05) when administered orally at 250 mg/kg and 500 mg/kg. The dry powdered leaf was found to contain alkaloids, glycosides, saponins and tannins. It is revealed from the screening model used that the methanol extract and aqueous fraction of this plant possesses acute anti-inflammatory activity.
Introduction
The use of plants to treat ailments is as old as antiquity. Records of humans using plants to treat diseases have been recorded as far back as 6000 to 4000 years ago when Ayurvedic physicians started treating tumors with extracts from Vinca roseus [1]. The use of medicinal herb in the treatment and prevention of diseases is attracting attention by scientists world wide [2]. The use of traditional medicine and medicinal plants in most developing countries, as a normotive basis for the maintenance of good health, has been widely observed [3].
Anthocleista djalonensis commonly called “Fartanlafla” in Mali [4], “Osuo” in Bayelsa (Southern Nigeria) is used traditionally for the treatment of various diseases, the plant is known for antipyretic, stomachic, analgesic and purgative actions [56], the aqueous extract was reported to produce a rise in blood pressure of cats and an increase in tone and amplitude of movement of rabbit duodenal preparations. The root decoction has been used in the treatment of diabetes mellitus. Herbalists claim a high percentage of cures in their diabetic patients treated with it [7].
Traditionally, the leaves are reputed to be used for the treatment of malaria and jaundice [8]. The bark is used as a purgative in small doses as large doses are considered toxic. According to the Mendi ethnomedicine, when the tree is used as firewood, the people sitting around the fire become sick [9].
The decoction of the leaves is drunk in Sierra Leone as treatment for jaundice, in Ivory Coast the root is used as a diuretic, vigorous purgative, poison antidote, treatment for leprosy, as an emmenagogue and in the treatment of edemas and elephantiasis of the scrotum. The root decoction is taken against chest pain, constipation and stomach pain [10]. The study was therefore aimed at investigating the anti-inflammatory activity of the leaf extracts with a view to justifying the use of the plant in the treatment of oedemas and elephantiasis of the scrotum.
Materials and Methods
Plant collection and identification
The fresh leaves of Anthocleista djalonensis were collected in Ugbowo area of Benin city, Edo State, Nigeria, in March 2008 and identified and authenticated by Dr. B.A Ayinde of the Department of Pharmacognosy, University of Benin, Benin City, Nigeria, were a voucher specimen has been deposited. Dried leaves of Anthocleista djalonensis were reduced to a fine powder with a mechanical grinder, filtered, and the fine powder was stored in a non-toxic polyethylene bag.
Extraction and fractionation of plant
The fine powder (600 g) was marcerated in 2.5 litres methanol for 48 hours, filtered, concentrated in a rotary evaporator, weighed (84 g) and stored. Forty grams of the concentrate was dissolved in methanol and successively and exhaustively partitioned into various fractions using n-hexane, chloroform and water. The various fractions were evaporated to dryness, weighed and stored in the fridge.
Phytochemical screening
Qualitative assay, for the presence of plant secondary metabolites such as carbohydrate, alkaloids, glycosides, flavonoids, tannins and saponins were carried out on the powdered leaves following standard procedure [11].
Drugs and chemicals
Indomethacin (BDH chemical), n-hexane (sigma-Aldrich), chloroform (Sigma-Aldrich), methanol (Sigma-Aldrich), ethylacetate (BDH-chemicals), and normal saline (Unique pharmaceuticals).
Pharmacological evaluation
Animals
Albino rats (170 – 200 g) of both sexes were kept at the laboratory animal house of the Department of Pharmacology and Toxicology, University of Benin, Benin City. Nigeria. The animals were maintained under standard environmental conditions and were allowed free access to feed (Bendel Feeds and Floor mill, Ewu, Edo State, Nigeria) and water ad libitum. All the procedures were conducted in accordance with the guide line for Care and Use of Laboratory Animals published by the National Institutes of Health.
Determination of Anti-inflammatory Activity
The albino rats were divided into groups of 5 each. Acute inflammation was induced by intra-plantar administration of 0.1ml 1% freshly prepared Lambda-carrageenan or dextran solution into the right hind paw of each rat [1213].The rats were treated with either normal saline, crude extract (250 mg/kg or 500 mg /kg P.O) or aqueous fraction (250 mg/kg or 500 mg/kg P.O) 1 hour before administration of phlogistic agents. Paw volume of rats were measured prior to administration of phlogistic agents and then at predetermined intervals. For carrageenan the interval was 1 hour for 6 hours while dextran was 30 minutes for 4 hours, change in paw volume was measured using Vernier calipers and anti-inflammatory activity calculated.
Statistical analysis
Results were expressed as mean ± standard error of mean. Statistical analysis of the data was done using one-way analysis of variance (ANOVA) followed by Dunnett’s test and significance determined using P-values < 0.05.
Results
Table 1: Effect of methanol extract of Anthocleista djalonensis on Carrageenan-induced paw edema in Rats
Table 2: Effect of aqueous fraction of the methanol extract of Anthocleista djalonensis on Carrageenan-induced paw edema in Rats
Table 3: Effect of methanol extract of Anthocleista djalonensis on Dextran-induced paw edema in Rats
Table 4: Effect of aqueous fraction of the methanol extract of Anthocleista djalonensis on Dextran-induced paw edema in Rats
Table 5: Phytochemical screening
Discussion
Inflammation is the response of living tissues to injury. It involves a complex array of enzyme activation, mediator release fluid extravasation, cell migration, tissue breakdown and repair [14]. It is also known that anti-inflammatory effects can be elicited by a variety of chemical agents and that there is little correlation between their pharmacological activity and chemical structure [15].This associated with the complexity of the inflammatory process makes the use of different experimental models essential when conducting pharmacological trials.
The present study establishes the anti-inflammatory activity of the methanol extract (crude) of Anthocleista djalonensis and the aqueous fraction of the crude extract in a number of experimental models. Carrageenan induced rat paw edema is a suitable experimental animal model for evaluating the anti-edematous effect of natural products [16] and this is believed to be triphasic, the first phase (1hr after carrageenan challenge) involves the release of serotonin and histamine from mast cells, the second phase (2hr) is provided by kinins and the third phase (3hr) is mediated by prostaglandins, the cycloxygenase products and lipoxygenase products [17]. The metabolites of arachidonic acid formed via the cycloxygenase and lipoxygenase pathways represent two important classes of inflammatory mediators, prostaglandins (products of the cycloxygenase pathway) especially prostaglandin E2 is known to cause or enhance the cardinal signs of inflammation, similarly, leukotriene B4 (product of lipoxygenase pathway) is a mediator of leukocyte activation in the inflammatory cascade [18]. From the results, the methanol extract of A. djalonensis leaves inhibited Carrageenan induced rat paw edema at 250 mg/kg and 500 mg/kg, although not significantly different (P> 0.05) from the control (table 1).The aqueous fraction of the methanol extract significantly inhibited (p < 0.05) carrageenan induced rat paw edema, at the third hour the activity of the aqueous fraction is higher than that elicited by Indomethacin in a dose-dependent manner. Indomethacin is a cycloxygenase inhibitor, the methanol extract has activity which is comparable to Indomethacin and can be said to inhibit the cycloxygenase enzyme but lipoxygenase inhibitors also possess significant anti-inflammatory activity against carrageenan induced paw edema [19], so inhibition of carrageenan induced paw edema by the crude extract could also be due to its inhibitory activity on the lipoxygenase enzyme.
Dextran induced edema is a well known experimental model in which the edema is a consequence of liberation of histamine and serotonin from mast cells [20]. At 500 mg/kg the methanol extract significantly inhibited dextran induced rat paw edema (P<0.01) after 60 minutes (table 3). The aqueous fraction also inhibited dextran induced edema significantly (P< 0.05) both at 250 mg/kg and 500 mg/kg after 60 minutes (table 4).
The methanol extract showed a dose dependent activity but was less than that produced by Indomethacin (table 2), the aqueous fraction showed an improved activity as compared to the methanol extract with a maximal effect at 500 mg/kg which was greater than the effect produced by Indomethacin after 60 minutes (table 4).
Preliminary phytochemical screening shows that the extract contains alkaloids, glycosides, saponins and tannins, it has been reported to contain phthalides, xanthones, glucosides and steroids [21].
Conclusion
It can be concluded that both the methanol extract and aqueous fractions of the methanol extract of Anthocleista djalonensis has anti-inflammatory activity against carrageenan and dextran induced paw edema in rats. These activities may be due to their content of tannins, alkaloids, glycosides, saponins and glucosides.
The aqueous fraction of the methanol extract showed better activity profile compared to the methanol extract hence it can be said to possess majority of the activity.
This study demonstrates the efficacy of Anthocleista djalonensis as an anti-inflammatory agent and also scientifically justifies the use of this plant as an anti-edematous agent in folk medicine [22], however, further studies are required to determine the constituents responsible for its anti-inflammatory activity and further authenticate its mechanism of action.
Acknowledgement
The authors are grateful to Dr. B.A Ayinde for his technical assistance and Mrs B.D Okunrobo for reading through the manuscript for it syntactic errors
