I have aqueous and ethanolic extracts which I plan to reconstitute in PBS and DMSO before doing enzyme inhibition assays on alpha amylase and alpha glucosidase. I will need to do serial dilutions to determine the minimum inhibitory concentration of each extract, but what should I use as the starting concentration of the crude extract?
This is a very broad-based question! I think a good approach would be to choose a positive control. Look for a known (commercial) inhbitor(s) of these enzymes and use the reported Minimum Inhibition concentration of the known inhibitor as the starting point (concentration) for your extract. Based on the preliminary result you can use increasing concentrations of the extract (if the extract is less inhibitory than the positive control) or serial dilution of the extract (if the extract is more inhibitory than the positive control).
You can make a comparative study between the positive control and your extract to get an ideal how good the extract is in enzyme inhibition
Good luck
Your objective is to simply to determine the minimum inhibitory concentration required. The inhibitory potentiality would depend on the nature of the putative inhibitor. The carbohydrases that you allude to respond broadly to inhibitors. Is your ethanolic extract reconstituted from evaporated material? The assessment could be done as follows, just as in any enzyme determination: starting from 5 uL of the extract and up. Five or six different concentrations could be selected. Tis would facilitate the potential estimation of the concentration needed for 50% inhibition (and the determination of IC50values), of which you are not seeking. . Since the time course of the reaction is not known, you need to repeat the assays for different time periods, subsequent to your determination of the "minimal" active concentration. Sine you would be looking to arrive at minimal inhibitory concentrations, the concentration of DMSO would also be low, and may not impair enzyme activity as such. For alpha-glucosidase , acarbose may be employed as a as the reference inhibitor, which would also aid in assessing the influence of DMSO on the enzyme.
Best!
Thank you, Dr. Govindaraghavan and Dr. Kandaswami. I will use acarbose as the positive control. Any suggestions of where I may find the MIC value for Acarbose? I have tried the Bayer website and Google Scholar searches, but have not had any success.
Check this link. This gives an IC50 value (while using as a positive control) for acarbose. Hope this is useful.
http://www.actabp.pl/pdf/2_2008/391.pdf
First, I normally make a stock solution with the highest concentration as possible eg, 50 mg'mL or 20 mg/mL. For assay, I ususally start with 200 mcg/mL as the max conc. I'm not sure this help or not. I'm doing anti-cancer medicinal plant extracts.
Hello Dr. Lumlerdkij, thank you also for responding. May I ask how do you decide between 50 or 20 mg per mL? Since you are doing anti-cancer research, I imagine that you are trying to find extracts that are toxic to cancer cell lines. Perhaps these concentrations may be too low for enzyme activity?
Dr. Govindaraghavan: Thank you for the article you sent, it contains critical steps including the starting concentrations of the serial dilutions for the extracts.
Dr. Lumlerdkij, the range of concentrations mentioned in the article, started at 62.5mg/mL and ended at 1.95mg/mL so the concentrations that you suggested are within that range.
Thank you both very much.
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