Freshly harvested leaves sample of Ageratum houstonionum were dried under
shade and powdered. Leaf sample of A. houstonionum was extracted by process of
hydrodistillation using a Clevenger-type apparatus for the preparation of essential
oil. Extract from A. houstonianum was prepared by dissolving 5 μL of the essential
oil in 10 mL methanol. All the sample was filtered through a Whatman (Maidstone,
England) stainless steel syringe assembly using a 0.22 μm Durapore (Millipore:
Milford, USA) membrane filter. Purification processes via column chromatography,
thin layer chromatography and preparative thin layer chromatography were done.
Reverse phase 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 ×
4.6 mm i.d.; 4 μm), a model 490 E 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.