I have been conducting an alpha-amylase inhibitory activity assay using the DNSA (3,5-di-nitrosalicylic acid) method with Acarbose as the standard inhibitor. The absorbance values obtained for Acarbose exhibit the expected trend of decreasing absorbance with increasing concentrations, indicating inhibition of alpha-amylase activity. However, when testing sample extracts, the absorbance values show an unexpected trend of decreasing absorbance from low to high concentrations. we tried passing the extract through charcoal as well, yet the trend is not changing. could anyone please recommend modifications for the DNSA method or possibly a different working protocol to carry OUT the experiment. THANK YOU.
Could you clarify your experiment a little more? What is the source of the amylase in this experiment? What are the "sample extracts" and what are you expecting to be in them? What is the a priori hypothesis you are testing?
It sounds to me like the acarbose is a control experiment and you confirm amylase inhibition (i.e. decreased enzymatic rate with increased acarbose).
The "sample extracts" you expect to contain natural product inhibitors of amylase? Or they are the source of naturally derived amylases that you are testing inhibitors on?
To get at the root of your question, there are other assay methods that might be useful, in particular continuous colorimetric methods that don't require stopped reactions with extended heating like DNSA does. If you provide some more details I would gladly offer my suggestions.
Cheers,
ACA
Thank you, Mr. Alexander for your response. We used porcine pancreatic amylase for our experiment. Acarbose was used as a positive control where it exhibited decreased enzymatic activity with increased concentrations. The extracts are ethyl acetate fraction of plant samples which are traditionally used in the treatment of Diabetes. We are attempting to check the antidiabetic activity of the extracts on Alpha-amylase in comparison to the standard (Acarbose). But then, the trend that has been observed for Acarbose was reversed in the case of samples giving higher enzymatic activity at higher concentrations. Please give your inputs with regard to this. Thank you.
Good day Chandana Mary Jasti,
From what you explained above, the reduced enzymatic activity with increasing Acarbose concentrations suggests appropriateness of the test protocol employed.
However, ethyl acetate (which is unlike used for extraction in traditional medicine) may not have extracted the inhibitory compounds. Also, the scientific basis for the traditional antidiabetic use of your research plant may be via mechanisms other than pancreatic amylase inhibition.
For the trend observed for Acarbose which was reversed in the case of the plant sample giving higher enzymatic activity at higher concentrations, the extract should be checked for carbohydrates/reducing sugars to rule out the possibility of concentration-dependent increase in substrate/product amount in the plant sample reaction tubes instead of fixed amount of starch used..
Thank you.
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