I am a researcher working on DPPH-(2,2-diphenhlpicrylhydrazyl) antioxidant assay of different extracts of the plant Cassis tora. Though there are positive results mentioned in the literature, I am not getting them even after repeating the test. Could anyone kindly suggest to me what could be the possible reason/s for this?
One of the first reasons that comes to mind is the quality of the DPPH reagent, and then the quality of the DPPH solution. I also ask if the methodology used is the right one? It could also be that the waiting time is not sufficient to allow for a detectable reaction
There are several possible reasons for this.
1. The DPPH protocol you are using may be incorrect. Or your DPPH chemical may be corrupted. You can test the DPPH procedure you used with a positive control such as trolox or ascorbic acid.
2. There may be errors in the preparation of the plant extract. Or the samples you used may have deteriorated due to storage conditions. You should make sure that the plant extracts you use are fresh.
3. The samples you use may be intensely colored. Therefore, they may give incorrect results. In this case, you can dilute your samples more.
Good luck
Please check the absorbance (OD) of the DPPH alcoholic solution prepared.
1. Please try to prepare the reagent in dark and set the OD before the experiment.
2. The absorbance is usually 1±0.1
3. Also, the OD of the samples (plant extracts) should be less than the DPPH solution.
4. You can crosscheck your results by comparing it with some standards (Ascorbic acid, BHT, etc.)
I hope it may help you getting your desired results.
All the best.
this could happen because
1. solvent extraction. the majority of compounds with antioxidant activity are polar. so it must be extracted with polar solvents, such as methanol, ethanol, water etc
2. If you make a series of solutions and there is still some extract that has not dissolved, do sonication. Don't filter it, because the solution content will definitely be much smaller than expected
3. Insufficient sample preparation. If you make a small amount of extract using a micropipette. Errors in using a micropipette can affect the levels in the solution, thereby affecting its activity
My suggestion is to make a DPPH solution of 40 ppm, and an extract solution of 1000 ppm. take 2ml DPPH and add 1 ml extract solution. If within 30 minutes there is a decrease in the intensity of the DPPH color, then it is likely that your extract has antioxidant activity. if there is no or little change, you need to make a new extract
Probable Reasons could be
1. The plant extract being used might not have significant free radical scavenging potency however, this is rare as many phytocompounds do exhibit good results in in vitro antioxidant assays such as DPPH and FRAP.
2. There could be some problem in the retrieval of the active constituents during extraction procedure, the factors may include choice of solvent used for retrieval and extraction methodology. It is better to use lyophilized end products for assessment of antioxidant activity and during DPPH assay please try preparing the test samples in the parent solvent in which it is actually retrieved. Solvents with high polarity index like water, ethanol and n-butanol are preferrable if you are targetting for polar compounds.
3. DPPH soultion must always be prepared fresh and in dark conditions as light exposure nullifies its effects. Methanol is used to prepare DPPH solution.
4. Time is an important parameter in the reaction of DPPH with the assumed antioxidant since it is a kinetic driven process. An incubation period of half and to one hour maximum is the standard time for the reaction to take place. You can test varying concentrations of the plant extract in two fold serial dilution manner to check its efficacy. This will address your concern. You can prepare the dilutions from 1mg/ml stock concentration, 5 different set of concentrations ranging between (10,000 μg/ml- 6.25 μg/ml) will do. Also prepare another set of standard using Ascorbic or gallic acid. This will rectify the mistaken step. If you will get the results in standard that means the methodology is fine and there is some issue in the test samples
5. It is always preferrable to prepare all the soultions on the same day of experiment.
6. Calibrate the spectrophotometer before the OD readings and take the readings at 515nm. A systematic decrease in absorbance from lower to higher concentrations will indicate good scavenging dose dependent antioxidant activity. With increase in concentration of antioxidant, DPPH absorbance will decraese.
7. Measure the EC50 effective concentration by plotting a graph with concentrations on y axis and percent inhibition on x axis. It is non linear.
8. Use only methanol as blank and control is prepared using DPPH and parent solvent used for the extract. Also please try using milli q water if required.
9. Make sure that the sample concentrations used must not be greater than the concentration of the DPPH solution
HOPE THIS HELPS
GOOD LUCK
I think that several factors can contribute to the differences between your results and the literature. For example, these may include storage conditions, extraction method, or solvent used, and you need to optimize the protocol before starting your manipulation.
You should also bear in mind that the collection period, geographical origin, and part of the plant (leaves, stems, roots) can lead to variations in the result.
I suggest trying again with DPPH and ABTS or reducing power to be sure.
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