am trying to do some extracts of metabolites from fungi to evaluate for antibacterial properties. I am going to do an ethyl acetate extraction. If I want to do a methanol extraction, do I do that before or after? do I use the ethyl acetate extract and then extract with methanol (I don't have experience with this so I need detailed protocols). I appreciate your help. thanks
Dear Olga,
You start the extraction with ethyl acetate and then you extract with methanol.
I have copid some important paragraph from a recent review (attached) which will be helpful to select your isolation method:
The majority of isolation procedures still utilize simple
extraction procedures with organic solvents of different
polarity, water and their mixtures.1,71,72 The methods include
maceration, percolation, Soxhlet extraction, ultrasound-assisted
extraction and turbo-extraction. Maceration is carried out
at room temperature by soaking the material with the solvent
with eventual stirring. It has the advantage of moderate
extraction conditions but suffers from high solvent
consumption, long extraction times and low extraction yields.
Extraction yield is improved by percolation, i.e. packing the
pre-soaked plant material in a container which allows the
constantly controlled removal of the extract via a valve at the
bottom and adding fresh solvent from the top. Soxhlet
extraction is a popular method for extraction due to its
reduced solvent consumption; however, thermo-labile
compounds might be degraded during the extraction process.
For liquid samples extraction by organic solvents or heterogeneous
solvent mixtures can be done, either simply in a
separating funnel or similar to a Soxhlet apparatus in a
perforator. On a smaller scale, extraction of the liquid sample
absorbed on a porous matrix (like diatomaceous earth) packed
in a column with non-miscible solvents is an option (e.g.
Extrelut columns).
Hoping this will be helpful,
Rafik
If you have grown the fungus in submerged culture, then you first separate the mycelium from the broth by filtration or centrifugation. The mycelium is treated with methanol to extract the metabolites. If you wish you can add ethyl acetate after the methanol extract is removed.
The supernatent can be tested in wells or on disks. You can extract the supernatent with ethyl acetate, but if the active compound is not extractable with ethyl acetate you can use an adsorbent resin (XAD 4 or 7) or ion exchange resin to 'extract' the active compound.
The suggestions mentioned above may be right but keep in mind that it all depends upon the characteristics of the intended metabolites in the living tissues . For the aqueous systems in the fungi , the Aqueous methanol will be best solution to bring the metabolites in the extract . Now extract with Ethyl acetate solvent ,evaporate this extract to very small volume, few ml , perform the TLC test on a square type ( 4 X 4 inches size ) to check first on a one dimensional side , the behaviour of the extract contents , the same plate can be eluted on the second dimension . this will show us the no / amount of the impurities being eluted in the first dimension. The solvent system we just have to find out through the trial basis & use that which gives maximum no. of compounds on the TLC plate. The Column chromatography solvent system has to be fairly stronger due to thickness of the silica bed or cellulose column bed . Collect the column fractions & do their UV , try to separate them as per their behaviour. Separate the one U R interested most , evaporate them for further study & chemical characterizations .
In practice the ethyl acetate extract is more effective than metanol extract in inhibition of microorganism growth, most of all.
Dear Zahra , Olgas first aim is to have the proper & complete extraction of the Metabolites from the Fungus Cultures . Once She has the extract , then only she can do the Antibiotic Activity assays . The answers given above will resolve her objective for the purpose. Dr Abid Aziz Ajaz
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