Is there anyone knows advantages and disadvantages of using bioassay guided fractionation/isolation method? Thank you.
Hi, Do you want to isolate the active compound(s)? If yes, I recommend you to do metabolite profiling of your fractions/raw extracts by LC HR MS/MS and bio-activity studies, than by using multivariate statistic (PLS DA), I think you can determine the active compounds without any isolation processes. After this, if you want you can isolate the active compound(s)
Hi, these are based on my and my friends experience in the labs because research is fun and curious at the same time.
Pros:
1. You can get active compounds, sometimes the most active that is responsible for the activity of your extract,
2. You can find and develop methods for the isolation of active compounds from the extract. This will be a protocol for you and team and a secret recipe to other researchers once you published the work.
Cons:
1. The process to get the active compounds may take time and exhaustive efforts than the non-bioassay-guided isolation as you will do test the activity of each fraction from chromatography works, every single fraction to trace the active one,
2. The active may present in a minute amount (sometimes 1 mg or less for spectroscopic measurements and assay).
Dear Dai Chuan Tan ,
Cons:
- If the activity evidenced in the crude is as a result of synergy then bioassay-guided isolation will be a wild goose chase.
- Depending on your protocol, the active compound might get adsorbed to the stationary phase.
- Above all, its time consuming!
Bioassay guided fractionation is useful to find the actual active compound from an extract. The bioassay is acting as a very specific detector.
The major disadvantage it the time to get results; depending on the bioassay, this ranges from several hours to months. There is also the time required for sample prep so the solvents from the chromatography don't interfere with the assay. There could be a lot of fractions! Tannins from plants often interfere with some peptide binding assays, but these show usually as non-specific binding across may assays. The azide preservative in some resins also sometimes cause a false positive.
I do recommend a literature search on the extract source (such as the plant species) and seeing if the known compounds are present and contribute to the assay activity.
Bio-guided isolation can be very helpful to determine the active fractions of extract, however the following points should be considered:
1-The dereplication of new compounds from this method could be very challenging,(In many cases the isolated compounds is known which is incompatible in case of the target is to obtain a patent.
Solution: Metabolomics/chemometric and neural network can reveal the presence of new compounds that can't be detected by traditional methods.
2-The choice of the assay may affect the results: negative results does not means that these fractions isn't active, if a certain plant is reported to possess anti-inflammatory properties, the chosen assay should be general rather than target based(such as COX-2 inhibition assay) since we don't know how the constituents exert their pharmacological effect so if the compounds are not inhibitor for COX-2 the results will be negative leading to discarding a possible active compounds.
3-In-silico tools could be integrated to predict the possible mechanism of action of the compounds so that a proper assay could be used.
Hi Prof. Gunawan Indrayanto, yes i’m isolating active compound. Currently i am doing NMR metabolomic
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