Two possible explanations are:

• The active compound has not left the HPLC column. If the biotest is sensitive to particles, the filter effect of the HPLC column might also account.
• Synergy: The activity depends on two or more substances which have been seperated by HPLC. Try if a mixture of your HPLC fractions has activity.

Two possible explanations are:

• The active compound has not left the HPLC column. If the biotest is sensitive to particles, the filter effect of the HPLC column might also account.
• Synergy: The activity depends on two or more substances which have been seperated by HPLC. Try if a mixture of your HPLC fractions has activity.

There are also 2 other possibilities.  Some enzymes require both cofactors and coenzymes to be active.  A cofactor, as an example, would be Calcium, while a coenzyme would be NADHP or ATP.

You may have separated them apart by HPLC.  It depends on the enzyme.

There is more going on with purification than just separation. If the purified material was taken to dryness, activity may not have resolubilized in your chosen solvent without being part of the original mixture. Or, activity could be due to mixed activity of weakly active substances. Or, the active material tailed off the column and you have not collected it.  Even though you identify a clear peak, that peak may not be the active substance. Or, the activity oxidized, decomposed, with purification away from other material.

How did you purify your compound up to HPLC? How did you dry it afterwards? This gives us a sense of the compound polarity, and how it could elute from HPLC. What solvents did you use to elute from the HPLC? Did you run a wash step?

Try washing your HPLC column with 100% acetonitrile and follow that up with dichloromethane (silica based column only!!!). Collect these fractions and then wash the column again with acetonitrile (so it can be used with water again). This procedure washes off very non-polar compounds.

The compound may have come off in a broad and dilute peak, so a modifier may be needed.

The compound may have decomposed during drying, and a freeze-dryer may be a better choice.

Yep, it's easy to come up with possible explanations, but harder to test and modify. The previous answer by Jack begins to offer ways forward.  What solvents can you use to resolubilize the purified material. What solvents will your activity assay tolerate? Can you use a different type of column with different solvents and ways to strip the column. Could you dry with a stream of nitrogen and then quickly resolubilize? Could you mix all the eluted fractions, dry, and obtain the activity back?  Is the activity very hydrophobic or very hydrophilic? Can you stick it to ion exchange and get the activity back? Can you estimate the potency of a semi purified material that maintains activity? How complex is the active semi purified material by NMR or mass spec or analytical hplc? Did the hplc elution solvents change the pH and lead to inactivity or decomposition?

It’ss possible that often certain molecules act by synergy, such as for example the fact of allosteric enzymes.

thank you for your comments and advice. 

The solvents and columns used for column chromatography (silica gel with non-polar and non-reactive solvents, GPC, etc) and HPLC (polar solvents typically with acid modifier and reactive organics such as MeOH).  One very probably cause is that the column purification used a non-nucleophilic solvent, so the compound survived and still showed activity. 

We often find metabolites that do not survive purification by HPLC.  Some compounds hydrolyze rapidly in acid while others are more stable in acid.  Further, many compounds will methylate in MeOH.  Another issue frequently encountered with HPLC fractions is that evaporations of these fractions will frequently use heat to remove water, and this will accelerate decompositions of many compounds.  Sonication in is often used to "redissolve" purified compounds, but this technique can also accelerate decomposition as well as denature enzymes.

Many solvents can also denature proteins, so this could be an issue as well.  Do you know if the active is a protein/peptide?  

Mr. Alshaibani,

So, you perform in vivo tests of a fractions (?!?) of secondary metabolites (meaning low molecular weight chemical compounds) without a complete separation of the ingredients followed after by test of presumably (?!?) separated compounds ('Presumably', because of HPLC also shows drawback toward complete separation).

Because of, the separation should be followed after with an unambiguous characterization of the structures of these ingredients, meaning their stable 3D structures under different selected set of experimental conditions; along with the obligatory tests for their chemical stability and reactivity (first, in an isolated state, but first because of there are second, third, etc.).

I agree with Jandirk Sendker, two main reasons: You have left the coumpounds on the column and moré than that the compounds act by sinergism. Good luck.

Flush the column with high polar solvents which bring out the compounds present on the column might give you the best activity.