As per my view, the dose which you have used for isolated extract might be low. You need to increase its dose and see if you get activity. Further if you isolated using column you should get more fractions according to increasing polarity. It might possible that activity will be shown in some other fractions which might not yet come from column.

Good luck

I mostly agree with Dr Carney's impressions. As an additional test to be conducted for evaluate any possible lost of components is to perform a TLC ( one or more) sampling the obtained fractions together with the crude extract. This could be of help for visualize eventual components not eluted or degraded during the work out.

You may also wash out the column with a suitable solvent to recover any retained component.

one or two spots do not mean you have one or two compounds, TLC is preliminary step and you can miss compounds which do not show UV Flo. Also it is impossible for you to characterize mixture of compounds by NMR, you did not tell us what kind of extract you have is it aq-methanolic or non-polar extract, may be you need to wash your column by abs-methanol. If you have good quantity of the extract try to purify all the compounds you can and test them separately, bio-guided assay has its own limitation like the occurrence of highly active molecule in low quantity (under the activity threshold) in an inactive fraction. 

Thank you for all the answer and suggestion. I appreciate it so much.

Yadu: We are looking for any possibilities of the natural-drug to have an activity at low concentration. But anyway, I might look at different test of bioactivity on this plant.

Carney & Venditti: I should use an appropriate word as "suggested" rather than "concluded". Yes, I have conducted the TLC for each elution by comparing with the crude extract. And all the fraction showed same spots with same Rf to the crude extracts.

Chaitanya: I'm looking towards to do a screening using GC-MS to see what phytochemical present in the plant. Then, I'm going to decide what is the next step.

Omar : It is 100% methanolic extract - polar. And yes, I have washed my column with an absolute of methanol. I'll try to purify those sample.

Synergism is not always the reason why crude extract maybe more bioactive than fractions. The questions to ask are: what is the dose of the fractions compared to that of the crude extract? What is the concentration of the active components in the crude extract compared to that of the fractions? The reason for the last question is because small amount of active ingredients may go into each fractions with resulting low activity.

Why don't you look back and check your extraction techniques. Instead of fractionating only crude methanolic extract, why don't you consider gradient extraction with solvent of different polarity. This what I mean by gradient extraction, extract same plant biomass with solvents of different polarity. Compare the bioactivity of each crude extract, then continue with fractionation of most active crude extract. This technique maybe more helpful in isolation of active components

What are your means of testing activity? If it's disc diffusion for the crude, you should use a more sensitive method for your purified/semi-purified samples (broth microdilution assay)

Thanks for your clarification

You need to think about macro-molecules which need special care for separation try to use Sephadex LH20, Methanol-Acetone if you have access to  LC-MS try to test your fractions to find at least molecular weights of your compounds; a point can help you to find a way

good luck   

Thank you for all the opinion. I'll try to check back my extraction method. =)... there must be some point that I missed out.