Hi, Sandeep -

A potential confounding factor is that the overall biological activity may be due to synergism between or among more than one substance, in which case individual fractions may show less activity than the original (complete) complex mixture. (On a related note, how are you measuring activity?)

To my mind, first of all you have to make the extracts of plant parts taken for evaluation. .You can make extracts in different solvents to get the detailed information about all the possible phytochemicals present in different extracts. These chemical compounds can be separated. After this each one of these can individually be tested for the activity, for example antidiabetic activity. Before this, you can try the extract as a whole just to check its activity. If it shows activity, then separate the different phytochemicals to check their specific activity.

Regards

First you qualitatively analyze the crude extract for presence and/or absence of specific groups of phytochemicals viz., steroids, alkaloids, phenolics, etc. Thereafter attempt separating different group of phytochemicals by the prescribed standard methods that include fractionation of crude extract with solvents of elutrope series as the first step followed by other compound specific methods. Now, carry out bioassay of all the fractions for specific pharmacological efficacy. Now the faction showing greater efficacy can be further resolved into pure individual compounds by chromatographic techniques like CC, HPLC, HPTLC, etc followed by bioassay of pure compounds. This is a multi-step chemical and biological experimentation to pin-point a particular compound for desired pharmacological activity.

On Annona squamosa - You should look at the number of papers by Prof. JL MacLaughlin on Annonaceae acetogenins. He has also reported acetogenins from A.squamosa bark. You should be able to lot of requisite information just by 'googling'

Regards

I agree with the above people that first collect different fractions of your extract by using different solvents and go for screening your activity. fractions which will show the activities will be further purified and characterized by different methods and that o depends upon your tye of compound which you want to extract.

I hope this will help you out

Hi, Sandeep -

A potential confounding factor is that the overall biological activity may be due to synergism between or among more than one substance, in which case individual fractions may show less activity than the original (complete) complex mixture. (On a related note, how are you measuring activity?)

The procedure is a bit complex(and u may need some modifications also, according to ur plant). First of all, make ethanolic, hydroethanolic and aqueous extract and checkwhich fracation is having activity.Then, go on with fractionation of the extract (not the crude plant) with descending grades of polarity viz, hexane, chloroform, butanol and water(u can refer the polarity index chart and substitute any of this with the solvent available with u).Then again check for biological activity with each fractions. Then go for column / flash chromatography to separate sub fractions from active fraction, which again need to be checked for biological activity. Then use Preparative (not analytical) HPLC for separation of compounds. find out active fraction. Then do GC_=-MS and or FTIR or NMR

Using a bioassay guided fractionation and isolation.

As per the valuable suggestions given by Ahmed Metwaly, John Wishnok and Yogesh Tripathi, it is evident that a bioactivity guided fractionation and isolation will be a help tool to know the elite molecule responsible for a particular therapeutic activity. Analytical Tools like TLC/HPTLC/CC/HPLC/LC-MS/NMR/XRD will be helpful alonwith cell-line based assays. But we should also remeber that many times the entire efficacy of a particcular extract/fraction is due to the synergism shown by phytochemicals.

As rightly stated by many respondents, particularly Dr. John Wishnok, compounds in natural plant products have activity in a synergistic way. It may not be feasible to attribute a single compound to a particular activity. This is what makes the big difference between synthetic chemical compounds ( targeted towards a particular biological activity) and natural product compounds which work together to produce a known activity. The methods of isolating the compounds have been adequately addressed by the former respondents.I have a practical example of a product which was initially subjected to bio-assay fractionation and some pure and semi-pure compounds identified. I now subjected the fractions and compounds to a test for Natural Killer Cells Activation. Surprisingly, the crude extract, the pure and semi-pure compounds all show significant activity.

I am agree with all respondent's Dr, I think firstly you must make a phytochemical screening to this compound for investigating its structure i.e. is it has alkaloids, flavonoids or any other active compounds, after that try to isolate this compound or study the effect of the crude extract as a whole. The pharmacological screening must be done in vitro as well as on vivo in lab. animals as rodents . Acute and chronic toxicity are also very important study especially LD50 . The review data will help you in the biological activity i.e. indicate the work of the previous researchers . If the compound is chemical synthetically derivative or analog you can study and compare it with the activity of the parent compound. The pharmacological screening is well known but there is several tests and activities so the structure and review articles are helpful guide for us, from what we must begin?

Hi Sandeep

First u have to collect extract with suitable solvent then evaluate its biological potential, after that partitioned the extract with solvent of different polarity to have different fraction then further evaluate their individual biological activity, out of different fraction, the fraction which showed the maximum activity,will tell you the nature of compounds responsible for particular activity.

i.e. I was extracted plant material with methanol and then the biological potential of methanol extract was evaluated, further, this extract was partitioned with first hexane and then with chloroform, to have three fraction hexane, chloroform and pure methanol fraction. then biological potential of individual fraction was evaluated and it was found that the antioxidan activity of methanol fraction was higher than crude methanol extract where as that of chloroform fraction was lower than crude methanol extract. further hexane fraction showed negligible activity. these results showed that the increase in activity of methanol fraction in comparison to crude methanol extract may be due to increase concentration of phenolic acids, flavonoid and tannin. from the literature and further characterization of this fraction tells us nature of individual compounds which have been responsible for particular activity.