i have been doing cholinergic assay on natural product and for some of the samples, i get negative value for the inhibition activity. can anyone help me explain the meaning of the negative value results? im doing the assay according to the protocol below.
Sample screening for anticholinesterase inhibition activity
The cholinesterase inhibitor activity of the sample extracts will be evaluated by the modified method of Rahman and Choudhary (Atta-ur-Rahman & Choudhary, 2001). Briefly, 0.1M sodium phosphate buffer (pH 8.0, 150μL), test compound solution (10μL) and enzyme solution (0.1units/mL, 20 μL) will be mixed and incubated for 15 min at 25°C. 10μL of DTNB (10mM) then will be added and reaction will be initiated by the addition of substrate thiocholine 10μL, 14mM solution. The hydrolysis of the substrate can be measured by the formation of the coloured product 5-thio-2-nitrobenzoate anion formed by the reaction of DTNB and thiocholine, which is released by the hydrolysis of enzyme. The formation of the coloured product will be measured at 410 nm wave length after 10min. Galantamine at a final concentration of 10μM will be used as a positive control in the assay
Percentage inhibition will be calculated by using the equation:
Inhibition activity (%) = (1 − Absorbance of sample/Absorbance of control) × 100
Perhaps some of your extracts also absorb UV at 410 nm? That would make the "Absorbance of Sample" greater than the "Absorbance of control", leading to a number greater than 1. When subtracted from 1, you get a negative number.
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