Investigating the Trypanocidal Activity of Purified Precocene I by Reverse-Phase High-Performance Liquid Chromatography from Essential Oil of Ageratum houstonianum Aerial Parts | Chapter 16 | Technological Innovation in Pharmaceutical Research Vol. 10

 The leaves of Ageratum houstonianum are a common poisonous weed found in the Kangra Valley near Palampur, Himachal Pradesh, India. Ageratum houstonionum leaves were freshly collected, dried in the shade, and powdered. The essential oil from a leaf sample of A. houstonionum was extracted using a Clevenger-type apparatus and a hydrodistillation procedure. The essential oil of A. houstonianum was dissolved in 10 mL methanol to make the extract. A 0.22 m Durapore (Millipore: Milford, USA) membrane filter was used to filter the entire sample through a Whatman (Maidstone, England) stainless steel syringe assembly. Processes of purification using column chromatography The researchers used thin layer chromatography and preparative thin layer chromatography. A Waters HPLC system with model 510 and 515 pumps, a Rheodyne injector, a Novapak C18 column (250 x 4.6 mm i.d.; 4 m), a model 490E multi-channel detector, and a Millennium 2010 sata manager was used for reverse phase HPLC analysis. A Durapore 0.22 m membrane filter was used to filter the mobile phase elements. A linear gradient of acetonitrile:water (40:60) was used to elute pure acetonitrile in 60 minutes at a flow rate of 1 mL/min. At 210, 240, 280, and 320 nm, detection was made. The precocene was eluted in 25 minutes, the peak areas were consistent (average relative standard deviation was 0.78 percent), and the calibration curves (mass of precocene standard injected vs. peak area detected at 210 nm) were linear over the 0.05-10 g range (y = 6654454 x + 176626, r2 = 0.99 for precocene I and 4618457 x + 133472 for precocen In methanol, standard samples of precocene I (1 mg/mL) and precocene II (1 mg/mL) from Sigma (St Louis, MO, USA) were produced. Precocene has been discovered. On Vero cells cultured in Dulbecco's Modified Eagle Medium (DMEM) and supplemented with foetal calf serum (FCS) 20-40% at suitable conditions, I was tested for trypanocidal activity against Trypanosoma evansi. On Vero cells, in vitro cytotoxicity testing of precocene I at doses (1.56-100 g ml-1) was performed without FCS. Trypanocidal activity in vitro ranged from immobilisation, reduction, and killing of trypanosomes in ELISA plate wells. At 9 hours of incubation with 250 g ml-1 purified precocene I, there was a dramatic reduction in average mean trypanosome count to complete trypanosome killing (40.0.0 to 0.000.00). At 4 hours, this was statistically equivalent to diminazine aceturate (50 g ml-1). With a significant difference (P 0.05 to 0. 01), trypanosome counts dropped in a concentration and time-dependent manner. Purified precocene I and diminazine aceturate standard drug were cytotoxic to Vero cells at all concentrations except 6.25-1.56 g ml-1 and 1.56 g ml-1, respectively, in an in vitro cytotoxicity test. Higher anti-trypanosomal action was attributed to Precocene I. Precocene I could be a trypanocidal chemical for a new trypanocide in the near future. In addition to the in vitro approach, an in vivo test is required to confirm its full and robust trypanocidal activity potential.

Author (S) Details

P. Shaba
Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh (243 122), India.

N. N. Pandey
Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh (243 122), India.

O. P. Sharma
Indian Veterinary Research Institute, Regional Station, Palampur, Himachal Pradesh, (17) India.

N. N. Pandy
Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh (243 122), India.

J. R. Rao
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh (243 122), (Former), India.

S. Dey
Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh (243 122), India.

B. D. Mandal
Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh (243 122), India.

N. P. Kurade
Indian Veterinary Research Institute, Regional Station, Palampur, Himachal Pradesh, (17) India.

R. K. Singh
Indian Veterinary Research Institute, Regional Station, Mukteswar, Uttranchal, (263 138) India.

V. Bhanuprakash
Indian Veterinary Research Institute, Regional Station, Mukteswar, Uttranchal, (263 138) India.

P. Chaudary
Division of Bacteriology and Biotechnology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh (243 122), India.

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