ANXIOLYTIC PROPERTIES OF MITRACARPUS VILLOSUS (SW) DC.LEAF EXTRACTS IN MICE AND RATS
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ABSTRACT
The leaf of Mitracarpusvillosus (Sw.) DC. (Rubiaceae) is used in West African folk medicine for the management of a plethora of stress-related diseases including headaches and toothaches. In this study, preliminary phytochemical analysis of the ethylacetateleaf extract of Mitracarpusvillosus and its fractions were conducted. Vacuum liquid chromatography (VLC), column chromatography (CC) and thin layer chromatography (TLC) using solvents of varying polarities were employed to separate the constituents of the plant extract. The intraperitoneal and oral lethal doses (LD50) of the extracts were determined by Lorke‘s method in mice and rats. The sub-chronic toxicity studies were carried out on male and female Wistar albino rats by daily administration of ethylacetateleaf extract of Mitracarpusvillosus (312 – 1250 mg/kg) for 28 days during which body weight, feed and water consumption were monitored. After 28 days, effects of the extract on biochemical and haematological parameters were evaluated and the histological changes of the vital organs were also examined. The effect of the ethylacetateleaf extract of Mitracarpusvillosus on mouse behaviour was examined using various in-vivo models whichinclude the hole-board (exploratory behaviour), open field test (OFT), staircase test (SCT), light/dark box (LDB), elevated plus maze (EPM), diazepam-induced sleep and rota rod tests. The anti-nociceptive effects of the ethylacetate extract were tested on acetic acid-induced abdominal writhing, oro-facial formalin-induced pain as well as carageenaan–induced hyperalgesia in mice and rats. The effects of the extract on inflammation and body temperature were determined using formalin induced paw oedema and Baker‘s yeast induced pyrexia respectively in mice. The ethylacetateleaf extract of Mitracarpusvillosustested positive for alkaloids,steroids, terpenes, flavonoids, cardiac glycosides, resins and tannins while the biologically active fraction tested positive for terpenes and steroids. The intraperitoneal LD50 of the extract was calculated to be 1264.91 mg/kg and the oral LD50 was estimated to be greater than 5000 mg/kg in mice and rats, respectively. Ethylacetateleaf extract of Mitracarpusvillosus at doses of 312, 625 and 1250 mg/kg did not cause changes of food and water intake or body weight, but significant (p<0.001) increase of the weight of the liver and levels of the mean corpuscular volume (MCV) were recorded at 1250 mg/kg. Similarly, significant (p<0.05) changes in the levels of the hepatic enzyme alkaline phosphatase (ALP) and the renal index creatinine were recorded. There was no significant difference in levels of electrolytes (Na, K, Cl, HCO3) and urea. Histological evaluation of the organs presented with distortion of the structures of kidney and liver tissues at high doses of up to 1250 mg/kg. Graded doses of the extract (6.25 – 25 mg/kg) exhibited significant (p<0.001) anxiolytic effects by causing increase in locomotion and central square crossings in the open field test. Staircase test produced increase in locomotion and rearing. The percentage of time spent in the light compartment was prolonged both in the LDB and EPM tests when compared to control. At higher doses, the extract (100 – 400 mg/kg) significantly (p<0.01) and dose-dependently prolonged the duration of diazepam-induced sleep (p<0.05), decreased the number of both peripheral and central squares crossed in the OFT, decreased (p<0.001) number of head-dips in the hole-board test and reduced steps climbing in SCT (p<0.05) in mice. The most active fraction E2, exhibited a similar pattern of behavioural actions comparable to the crude extract. The ethylacetateleaf extract of Mitracarpusvillosusat the doses tested had no effect on motor co-ordination as observed in the rota-rod assay in mice. The extract at 100 – 400 mg/kg significantly (p< 0.05) and dose-dependently inhibited acetic acidinduced writhing, decreased the time of face rubbing induced by formalin in mice and ix increased the paw withdrawal threshold of carageenan induced hyperalgesia in rats. Paw thickness induced by formalin was also significantly (p<0.001) reduced. Hyperthermia induced by baker‘s yeast was significantly (p< 0.05) reversed by the extract. The actions exhibited by the ethylacetateleaf extract of Mitracarpusvillosus are probably mediated via the benzodiazepine-GABA-ergic (BDZ-GABA) pathways. The results from this study provide scientific evidence that the ethylacetate extract of Mitracarpusvillosus leaf may contain psychoactive principles that are sedative in nature with potential anxiolytic effects attributable to the presence of terpenoidal and steroidal compounds. The sedative and anxiolytic effects may be mediated through the benzodiazepine site of the GABAA receptor channel complex as the effect of the extract on diazepam induced sleep was reversed by flumazenil. The extract exhibited anti-nociceptive effects against neurogenic and inflammatory mediated pain with antiinflammatory and hypothermic effects. These findings support the further appraisal of the biologically active principles of the plant as analgesic, anxiolytic and antiinflammatory agents.
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