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• Anti-malarial Bioassay

Natural Products represent a rich source of biologically active compounds and have potential in drug discovery and development for Malaria. About 1200 plants from 160 families have been tested for determining their anti-malarial potential. Quinine from bark of Cinchona succiruba and Artimisinin from Artemisia annua are used as anti-malarial.

 

Method:  Parasite Lactate Dehydrogenase Assay (in-vitro)

Serial dilution of drug or test compound is exposed to a specific number of parasite ( Plasmodium falciparum 3D7). After incubation the effect of the compound was determined by calculating the LD50.  LDH activity is correlated with parasite growth and used as a marker of Plasmodium infection. The inhibition of LDH production by an increasing concentration of an anti-malarial drug is used as a measure to determine drug susceptibility pattern of malaria parasite.

 

Nature of Sample Tested:
Crude plant extracts and fractions.

 

Materials and Methods:

Plant extracts and standard drugs were serially diluted (two fold) with complete culture medium in 96 well plates. Infected RBCs solution with 2% parasitemia and 1% hematocrit was added making the total volume 200 µl in each well. Plates were incubated in candle jar with 5% CO2, 5%O2 and 90%N2 at 37º C for 72 hours. For malstat reaction, in 100 µl of malstat solution, 20 µl solution from each well of plate 1 to respective well of malstat plate was added. Plates were placed in shaking water bath at 37º C for 30 minutes. 25 µl solution (1:1 solution of NBT (2mg/ml) and PES (0.1mg/ml) was added in each well and the plates were kept in the dark for complete reaction and development of color. LDH of P. falciparum rapidly used 3-acetyl pyridine adenine di-nucleotide (APAD) as a coenzyme in the reaction leading to the formation of pyruvate from lactate. The conversion of NAD+ to NADH+H+ reduced the Tetrazolium to Formazan, purple color can be detected spectroscopically at 650nm and visually. The amount of color formed is proportional to number of parasites present. Plates were read at 650 nm and the OD was recorded. The % inhibition was calculated by the formula with Microsoft excel. % inhibition was calculated by the formula. IC50 was calculated with the help of EZfit computer program.