Depending on you EO's nature, you can try DMSO, which will not influence the antioxidant activity or dosages you mentionned. Ethanol and Methanol can also be used, do some blanc tests and adapt the concentration to your convenience.

Thank you Feknous Ines !

Feknous Ines can I ask about the EO concentration? let’s say the standard concentration I have done for calibration curve range from 200 to 2000 mg/L, does my sample (EO) concentration have to be in the range of standards or it does not matter?

Usually, we start with a concentration of 4mg/ml (4000mg/L) and perform a series of 7 dilutions. Then, according to the results you obtain you can adjust for the rest of the tests.

We start with the concentration of 2mg/ml for the standards (sometimes even 1mg/ml) because they have a proven strong antioxidant activity, and if you start from 4mg you'll need to perform a lot of dilutions to reach the IC50

Feknous Ines Is it okay to start with 1mg/ml for the EO concentration instead of 4mg/ml? or is the concentration too low? I am asking because I don’t think I can experimenting with the sample that much, due to the limited amount of it. unfortunately. And for the standards I have been using 1mg/ml. Thank you :)

Dear Farah Fatihah ,

Yes of course, you can even start with the EO concentration of 1mg/ml, it depends on both chemical composition and biological activities. If it's for antioxidant ones, try to work with standard equivalent instead of IC50 curves.

Good luck

Thank you Benabdallah Amina !!

@Farah Fatihah Yes, you can start with the concentration of your choice, it is up to you. Then, according to the results you obtain you'll adapt.

The first thing to do is to review the works related to your plant, to have an approximative estimation of where to start.

For the dosages it is simple, we don't need dilutions since in the protocoles we always take from the 1mg/ml sample concentration and perform it in triplicates (three repetitions)

For the ATox' activities, let's assume you started with 1mg/ml, prepared 6 dilutions out of it and analysed it. There are 3 possibilities:

1 : The absorbance values of your dilutions contain an interval where 1/2 initial absorbance of reagent is reached, which means you reached the IC50. You're good, no need to do anything else, and you have a good or very good ATox' activity

2: Even for your most diluted sample, the absorbance values are lower than 1/2 the initial absorbance of reagent, which means all the reagent was inhibited in the solution, you don't have the IC50 because your sample is too strong, you need to dilute it more and you have a very good to excellent ATox' activity.

3: Even for your most concentrated solution, your absorbance values are higher than 1/2 reagent absorbance, your sample is too weak and couldn't inhibit 50% of reagent. You'll need to start with more concentrated solutions.

Why do we look for 1/2 Initial absorbance of reagent? Simply because it represents the inhibition of 50% of it, which is the IC50. If I start with an ABTS adjusted to 0.700, my IC50 will be somewhere in the interval of absorbances that include 0.350

If you have any other questions or concerns, please feel free to DM me and we'll organise a visio meeting where we will discuss whatever you want in this area

Hi Feknous Ines

Thank you for the explanation. I will start using the EO sample next week and I do have some question regarding antioxidant assay. I will message you! ^^

Hello everyone in this thread.

Feknous Ines you just gave the most simple and detailed answer, an amazing read!