Assessment of the in vitro trypanocidal activity
Muhammad Muhsin Fathuddin
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Abstract
The aim of this experiment is to assess the potential of a plant extract to fight off Trypanosoma species, including Trypanosoma brucei brucei, Trypanosoma evansi , etc., by testing it at concentrations ranging from 006.25 to 400.00 mg/mL. The results obtained indicate that the plant extract may be a useful tool in the management of trypanosomiasis, but further studies are required to confirm its effectiveness.
Before start
The experimental animals should be screened for any ailment at the commencement of the experiment using the laboratory’s recommended procedure and cleared of any ailment.
Steps
Extract Preparation
To produce a stock Sample solution of100mg/mL for the extract, weigh1g (1000mg) of the extract, which was solubilized in 1 mL of dimethylsulfoxide (DMSO) solution and made up to 9mL in dextrose saline/normal saline.
Serial dilutions were made for the remaining concentration of50mg/mL, 25mg/mL, 12.5mg/mL and 6.25mg/mL of the extracts were made in Dextrose saline (freshly prepared).
Antitrypansomal Activity
The infected rat to undergo euthanasia must have attained a blood parasitemia of log 8.4 or higher
Euthanatized animal’s blood was dissolved in heparin (1mL of heparin per 10mL of blood) and mixed with glucose (0.1g of glucose per 10mL of blood).
Then, aseptically, a clean micropipette transfers the blood (50µL) to a clean, sterile microtiter plate into multiple wells.
To the two wells containing blood, the same volume (i.e., 50µL) of the highest drug concentration was added, respectively.
Step 4 was repeated for the other different concentrations available.
The negative control blood was mixed with the dilutant used to prepare the various concentrations (i.e., dextrose-saline or water).
The positive tests were also performed with the standard recommended concentrations
of a commercially available trypanocide drug.
At 0h 5m 0s 5-minute interval, a drop of blood is taken from each well and smeared on a clean glass slide. then observe under a microscope at 400x for signs of motility unique to Trypanosoma spp.
The above step is repeated with each well. Till motility stopped in all wells
