Well, there are two ways you can answer this question using a spectrophotometer;

The first one being Fluorescence quenching, depending on the enzyme that you use, the active site of the enzyme could contain a fluorophore. So by first taking a reading of the enzyme in a solution (buffer/water) by itself and then comparing the same enzyme in the presence of your inhibitor (quencher) you would be able to quantify the binding of the ligand (quencher) with the enzyme. Furthermore, by looking at the redshift or the blue shift of the fluorescence spectrum, you could find out whether the quencher has any influence on the structure of the enzyme at different concentrations.

The second way would be by conducting a simple reaction where the enzyme would act as a catalyst in the presence and the absence of an inhibitor. As an example, the enzyme alpha amylase catalyses the hydrolysis of starch to give you disaccharides and oligosaccharides, these products are reducing sugars which are able to bind with a colour reagent proportional to its concentration. The intensity of the colour would be measured using an absorbance test. Once you obtain a standard curve, you can then introduce your ligand/inhibitor, and the difference in the activity of the enzyme that you may observe would be due to the inhibition of the enzyme by the ligand. However, you must make sure to create proper blanks to rule out false positives.

I recently published a paper dealing with something similar, do have a look if you are interested.

Article Inhibition of starch digestion by flavonoids: Role of flavon...

Thank you

I think you are asking about how to tell if two or more different inhibitors in the essential oil are binding to the enzyme simultaneously in different sites.

To answer this question, you will have to fractionate the oil to purify the individual inhibitory components. This would probably be done using some type of chromatography. Individual fractions from the chromatography would then be tested for inhibition of the enzyme in an enzyme activity assay.

Once you have the individual inhibitors purified, you will be able to use steady-state kinetics, equilibrium binding, and/or structural biology to determine whether any pair of inhibitors can bind at the same time.

I was thinking of buying the main compounds of essential oil, for example for rosemary

pinene, camphene, limonene, cineole, borneol, camphor, linalool, terpineol, octanone and bornyl acetate.

FOR a chromatographic separation, which is not very easy to perform because it is necessary to find a suitable eluent mixture, once the compounds have been separated it is necessary to recognize them.

I docked natural amylase inhibitory compounds and found high scores in reference to acarbose.