Pharmacological Characterization of the Antiasthmatic Constituent of the Leaves of Asystasia Gangetica (L.) Anderson (ACANTHACEAE)

Abstract

Asthma is currently a worldwide problem, with increasing prevalence in both ?r children and adults; a prevalence rate of 5 - 10% has been reported for Nigeria. The pathophysiological features of the disease are bronchoconstriction, airway hyperresponsiveness and chronic inflammation. Drugs used in the management include bronchodilators which are short-term relievers and anti-inflammatory drugs which are long-term controllers. Despite the availability of oral and inhaled medications, the prevalence of asthma is on the rise. The challenge of developing b new effective, safe and long lasting antiasthmatic drugs from natural products appears inevitable. The leaves of Asystasia gangetica L. (T). Anderson sub species micrantha (Nees) Ensermu (Acanthaceae), a traditional anti-asthma remedy, offer ? great potential for the development of a novel anti-asthmatic agent. The leaves have been shown to possess antihistaminic, bronchospasmolytic and anti- inflammatory properties. The aim of this research was to isolate and pharmacologically characterize the anti-asthmatic constituent of the leaves of this plant using bioactivity-guided fractionation of the leaf extract. The leaves of A. gangetica were sun dried in open air for 48 h and pulverized to coarse powder. The leaf powder was extracted by cold maceration in 100% methanol for 48 h. The extract obtained was concentrated in a rotary evaporator under reduced pressure to afford the methanol extract (ME). The median lethal ?, dose (LDSd) of ME was determined in mice using the intraperitoneal route. The ME was fractionated in a silica gel (70 - 230 mesh) column successively eluted .. . Vlll with hexane: ethylacetate (7:3) and methanol (100%) to obtain fractions A, B and C. Screening of the three fractions for bronchospasmolytic activity using inhibition of histamine induced contraction of the guinea pig trachea and relaxation of pre-contracted trachea (pathological tissue) as bioactivity-guides showed that fraction B exhibited the greatest effect in both tests. Based on the results, fraction B was hrther separated using gradient solvent mixtures of hexane: ethyl acetate (9.5:0.5, 9:1, 8:2, 7:3, 6:4, 5:5, 0:l) to obtain eight fractions (Fr I, Fr 11, Fr 111, Fr IV, Fr V, Fr VI, Fr VII, Fr VIII). Activity-guided tests on these b fractions revealed greatest potency in Fr VIII, which on chromatographic separation successively eluted with petroleum ether: ethyl acetate (7:3) and ethylacetate (100%) yielded two fractions (Fr 1 and Fr 2). Fr 2 yielded a brown ' amorphous powder AG-1 which at 0.4 mg/ml completely inhibited the contractions of the guinea pig trachea induced by histamine. The methanol extract (ME), fraction B, Fr VIII and Fr 2 were subjected to phytochemical analysis for identification of constituents. The effect of the extract and fractions (200 and 400 mgkg) on systemic acute inflammation was studied in rats using the paw edema induced by carrageenan. Also, the effect of the extract and fractions (400 mg/kg) on carrageenan-induced leukocyte infiltration into the pleural cavity as well as pleural exudate formation was evaluated in rats. The data obtained was analysed using One Way Analysis of Variance in SPSS Version 1 1. + Phytochemical tests showed that the ME tested positive to alkaloids, glycosides, saponins, reducing sugars, terpenes and carbohydrate. Fraction B gave positive reactions for flavonoids and terpenes while Fr VIII tested positive to flavonoids, terpenes and steroids. Fr 2 gave positive reactions for terpenes and steroids. The 9 intraperitoneal median lethal dose (LDSo) of ME in mice was estimated to be greater than 5,000 mglkg. In the isolated tissue tests, the ME, Fr B, Fr VIII and AG-1 exhibited varying degrees of dose-dependent inhibition of contractions of the guinea pig trachea induced by histamine. The ME and the fractions also relaxed the guinea pig trachea pre-contracted with histamine. In the whole animal experiments, ME, Fr B and Fr VIII significantly (P<0.05) suppressed the b development of acute edema in the rat paw with peak effect at 1 h post induction of inflammation. In the rat pleurisy study, ME, Fr B and Fr VIII reduced the volume of pleural exudate with the effect of ME (400 mg/kg) and Fr VIII (400 P mglkg) being significant (Pc0.05). The ME, Fr B and Fr VIII reduced the total leukocyte count (TLC) with ME (400 mglkg) and Fr VIII (400 mglkg) causing a significant (P<0.05) reduction in TLC. The results established the anti-asthmatic potency of the leaves of A. gangetica and suggest that AG-1 may be the constituent of the leaves responsible for the anti-asthmatic action