Anti-trypanosomal activity of HPLC purified precocene I from Ageratum houstonianum leaves against Trypanosoma evansi

Shaba P; Rao J R; Sshab D; Tiwari R K; Singh R K

doi:10.21767/2348-9502-C1-006

Anti-trypanosomal activity of HPLC purified precocene I from Ageratum houstonianum leaves against Trypanosoma evansi

6^th Edition of International Conference on Pharmacognosy and Medicinal Plants
April 16-17, 2018 Amsterdam, Netherlands

Shaba P, Rao J R, Sshab D, Tiwari R K and Singh R K

Indian Veterinary Research Instituteâ�?�?Uttar Pradesh, India College of Agricultureâ�?�?Mokwa, Nigeria Indian Veterinary Research Instituteâ�?�?Uttaranchal, India

Posters & Accepted Abstracts: Am J Ethnomed

DOI: 10.21767/2348-9502-C1-006

Abstract

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 Clevengertype 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 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 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. In vitro cytotoxicity test of precocene I 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 I, there was drastic reduction of average mean trypanosomes count to complete killing of trypanosomes (40.±0.0 to 0.00±0.00) at 9 h of 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.