I have done a MTT cell proliferation assay on a cell line with 6 different concentrations of a plant crude extract, and replicated 4 times. In most cases, I found the activity was found in the lowest and highest concentrations together, which is statistically significant. How can the same activity be shown in two extreme opposite concentrations? Is there any explanation for this? Can anyone give any kind of evidence?
What are the result of MTT assay for intermediate concentration b/w two extreme range.
If same than Ok.
You can justify that drug concentration doesn't statically affect the Cell death till maximum concentration (X).
So try to find higher concentration which would statically show difference in MTT assay value.
I would recommended you also to check whether your methodology are well validated and precise.
If you share result (without disclosing your drugs and cell line) then someone would help you best.
You can concentration of drugs and MTT assay values for all and repeated experiment.
Hope you would understand well meaning of validated experiment well
my dichloromethane crude extract at 10^-5 g/ml and 10^-10 g/ml giving the activity which is in percentage is 140.4 % and 127.1% respectively (this is average value). The mthodology is well validated. the test is for cell proliferation not studying the cytotoxicity effect.
Dear , my suggestion is u have to try MTT starting from 100-1 microgram/ml.
Best regards
You need to deviate from the conc. vs efficacy concept here.
What you are using is a crude plant extract! This is not a single entitiy. It is a mixture of a multitude of compounds of diverse chemistries at different concentrations. This has to be borne in mind while interpreting the results.
To oversimplify..
1. Assume, there is one constituent that is effective against a target at a low concentration and a gradual increase in concentrations gradually reduces efficacy
2. On the same score, assume there is another constituent, which shows efficacy only at higher concentration
This would effectively answer your question. To prove or disprove this theory, you need to effectively monitor assays of different constituents at different dilutions/concentrations
and figure out which constituents correlate to activity.
You have plenty of examples for both 1 and 2 statements in systems biology.
A complex issue with use of crude extracts is - one needs to generate 'compound interactions' and the 'interaction effects'. You could have inter-play due to additivity, synergism and antagonism.
I think, the first step in understanding these complexities should be to 'deconvolute' the mixture - attempt an analytical HPLC and attempt a finger-print pattern; the look for correlations between concentrations of detectable constituents to efficacy data.
I know I am trying to over-simplify....This could at least be a starting point for your discussion.
All the best
Thank you very much Mr Suesh. excellent discussion and advise on this issue. will be very helpful for me.
MTT assay is a measure of how many living cells, but are you sure that the effects of the plant extracts against cell? inhibit proliferation or cause cell death. to ascertain the effect of inhibiting or enhancing proliferation, you should use a concentration that did not cause cell death. therefore, you must first determine how the concentration that causes cell death. then you can easily to determine how the concentrations used to measure the effect of proliferation.
Sir, Crude plant extracts are complex mixtures with multiple pharmacological activities. The different constituents could have antagonistic, allosteric and synergistic effects. At the higher concentration that was employed in your case, an active substance could have blunted the activity of a second bioactive constituent. It is not unusual to notice dual effects. In some cases, the putative component might be targeting different molecules or pathways (or different forms of receptors such as beta binding sites of estrogen) evoking cellular proliferation at different concentrations. In some cases, the activity of a component manifested at a certain concentration could have been modified at a different concentration owing to overriding of the original effect elicited. Some of the presumptive constituents might be redox active, and could display differential effects as well. Ascorbic acid and the dietary flavonol, quercetin, might engender divergent effects with different concentrations of a plant extract comprising them; one of them possesses a different redox potential. In certain instance, a potentially active agent, at a higher concentration, could be stabilized by a second component. Another factor to be considered with regard to the potential biologic activity of plant constituents is the oxygen environment of the experimental system, in this case neoplastic cells. You are assessing antiproliferative effect. Is there a cytotoxic/cytostatic effect shown by the plant extracts? Thanks…
Dear Chitan, thank you very much for your enlightening discussion. can you provide any literature that discusses this kind of issue you explained? I am not looking antiproliferative effect rather proliferative effect assuming my extract would stimulate the proliferation of the target neoplastic cells.
Dear Moyeenul, thank you kindly; I misread your question. Greetings.
Dear Moyeenul, one such example is that of the dual effects of an isoflavone, predominantly coming from soy; its biphasic effects on breast cell proliferation. I shall try to find the citation for you. L-Ascorbic acid is well known for ts opposite effects with low and high concentrations.
Thank you very much Chitan. that will be very helpful. I appreciate your effort for me.
With crude extracts first we have to look at the qualitative activity of the extract rather than quantitative activity.Some times the manipulation of the extract into small concentrations will be erroneous.First you interpret that the Extract possess activity.Now you try to fractionate the extract with 2-3 solvents and then try to find which fraction ismore active and follow with their TLC or HPTLC profile.
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