Dear sir/Mam

I have been trying to study the Xanthine oxidase inhibition of 1-ortho methyl chrysophanol isolated from actinobacteria. The formula was (A-B)-(C-D)/A-B X100. Here C is (Substrate+inhibitor+enzyme) and D is (Inhibitor+substrate) negative control. Here the problem is whenever I was performing the assay the OD value of negative control D is higher than C instead of C is higher than D. Could you explain what might be the reason. Or else explain me the correct procedure to study the XO inhibition of colored compounds (1 ortho methyl chrysophanol).

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