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Pharmacognosy 2018 American Journal of Ethnomedicine
ISSN: 2348-9502 Page 110
April 16-17, 2018
Amsterdam, Netherlands
6th Edition of International Conference on
Pharmacognosy and
Medicinal Plants
Freshly harvested leaves of Ageratum houstonianum were dried
under shade and powdered. Leaf sample of A. houstonianum
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. 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 1mL/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 (1mg/mL) and
precocene II (1 mg/mL) obtained from Sigma (St Louis, MO, USA)
were prepared in methanol. Identified precocene I was screened
against Trypanosoma evansi 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. InvitrocytotoxicitytestofprecoceneIatconcentrations
(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 I, there was drastic
reduction of average mean trypanosomes count to complete
killingof trypanosomes (40.±0.0 to0.00±0.00) at 9hof incubation,
which was statistically the same as diminazine aceturate (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)). During in vitro cytotoxicity test, Purified precocene I and
diminazine aceturate standard drug, were cytotoxic to Vero cells
at all concentrations except at concentrations of 6.25–1.56 µg
ml-1 and 1.56 µg ml-1, respectively. Precocene I was responsible
for higher anti-trypanosomal activity. Precocene I could be the
near future trypanocidal compound for a new trypanocide.
Anti-trypanosomal activity of HPLC purified precocene I from
Ageratum houstonianum leaves against Trypanosoma evansi
Shaba P1, 2, Rao J R1, Sshab D1, Tiwari R K 1 and Singh R K3
1Indian Veterinary Research Institute–Uttar Pradesh, India
2College of Agriculture–Mokwa, Nigeria
3Indian Veterinary Research Institute–Uttaranchal, India
Am J Ethnomed 2018, Volume 5
DOI: 10.21767/2348-9502-C1-006