Trypanosomosis, one of the neglected diseases, had resurged with debilitating to fatal effects on both animals and humans without a new drug to combat it. Freshly harvested leaves of Ageratum houstonionum were dried under shade and powdered. Extraction of the leaf sample of A. houstonionum was by the process of hydrodistillation using a Clevenger-type apparatus for the preparation of essential oil. Obtained extract of A. houstonianum was done by dissolving 5 µL of the essential oil in 10 mL methanol. The sample was filtered through a Whatman (Maidstone, England) stainless steel syringe assembly using a 0.22 µm Durapore (Millipore: Milford, USA) membrane filter. HPLC analysis was carried out via a Waters HPLC system consisting of model 510 and 515 pumps, a Rheodyne injector, a Novapak C₁₈ column (250 x 4.6 mm i.d.; 4 µm), a model 490E multi-channel detector and Millennium 2010 sata manager. The mobile phase constituents were filtered using a Durapore 0.22 µm membrane filter. The elution was carried out with a linear gradient of acetonitrile: water (40:60) to pure acetonitrile in 60 min at a flow rate of 1 mL/min. detection was at 210, 240, 280 and 320 nm. The precocene was eluted within 25 min, the peak areas showed good reproducibility (average relative standard deviation were 0.78%), and the calibration curves (i.e. mass of precocene standard injected vs. peak area detected at 210 nm) were linear over the range of 0.05-10 µg (for precocene I, y = 6654454 x + 176626, r² = 0.99 and for precocene II, y = 4618457 x + 133472, r² = 0.99). Standard sample containing precocene I (1 mg/mL) and precocene II (1 mg/mL) obtained from Sigma (St Louis, MO, USA) were prepared in methanol. Identified precocene II was screened against Trypanosomaevansi for trypanocidal activity on Vero cells grown in Dulbecco's Modified Eagle Medium (DMEM) and supplemented with foetal calf serum (FCS) 20-40% at appropriate conditions. In vitro cytotoxicity test of precocene II at concentrations (1.56-100 µg ml-1) was done on Vero cells but without FCS. In vitro trypanocidal activity varied from immobilization, reduction and to the killing of trypanosomes in corresponding ELISA plate wells. At 250 µg ml-1of purified precocene II, there was a drastic reduction of average mean trypanosomes count to complete killing of trypanosomes (40.±0.0 to 0.00±0.00) at 6 h of incubation, which was statistically the same as diminazineaceturate (50 µg ml-1) at 4 h. Trypanosomes counts decreased in concentration and time –dependent manner with significant difference (P ≤ 0.05 to 0. 01)). In in vitro cytotoxicity test, purified precocene II and diminazineaceturate standard drug were cytotoxic to Vero cells at all concentrations except at concentrations of 6.25-1.56 µg ml-1 for both respectively.  Trypanocidal activity recorded was due to precocene II. A source of a new trypanocide could be obtained from it.

KEYWORDS:  Ageratum houstonionum leaves, HPLC  purified Precocene II, In-vitro Trypanocidal Activity, ln-vitro Cytotoxicity Test.