|MadSci Network: Biochemistry|
1) Solvent fractionation or extraction is a process of separating various metabolites of an organism (plant, microbe, even animal tissues) based on their differential solubilities in different solvents such as water and organic solvents like acetone or hexane. Plants, for example, will naturally synthesize a great range of chemicals with a great range of properties. Solvent fractionation tends to separate based on hydrophobicity/hydrophilicity, which is basically how water-soluble (or not) the chemicals are. But with judicious choice of solvents, it can also separate on a more subtle basis such as by polarity. 2) There is no single “definite procedure,” but I will share some links for some procedures that I found online, as well as one of my own:
As a graduate student, I would routinely grow cultures of E. coli cells that made carotenoids, then extract the carotenoids from the cell pellets by a simple solvent fractionation technique. My thesis (the Materials and Methods sections of chapters 2 and 3) contains the details of what I did, and can be downloaded at http://resolver.caltech.edu/CaltechETD:etd-08232005-174620 . Basically, I exploited the fact that carotenoids were soluble in hexane but many other chemicals in the cells such as DNA, proteins, sugars, etc. were not, to do a fractionation with a hexane upper layer and a water (or salt-water) lower layer (hexane and water are immiscible), using a separatory funnel. This would allow me to concentrate the carotenoids in the hexane layer, which could be easily evaporated to dryness, and “clean” the cell extracts of other, water-soluble unwanted metabolites.
3) In my case and in many others, for example the second link above, further purification of the solvent-fractionated extracts is needed and is done through some type of chromatography. Without knowing the precise goal of your experiment, I can’t give you a number of fractions into which you will need to separate your extract for your project. Are you trying to narrow down to the specific chemical in the plant that has the antifungal activity? Or are you just interested in some basic properties of the antifungal compound, such as whether it is hydrophobic or hydrophilic? In general, I would think you would take a “divide and conquer” approach where you first separate the extract into hexane-soluble and water-soluble fractions, then test each for antifungal activity. Assuming only one of the fractions has the activity, you would then focus on that fraction and separate it further, then test the fractions derived from it for antifungal activity. Separation by chromatography will dramatically help you to narrow your search. I can’t tell you what specific type of chromatography to use but there must be a good deal of literature on indigo plant extraction.
(I found for you some specific papers on extraction of indigo leaves or similar tissues by Google searching for “solvent fractionation Indigofera leaf extracts;” I hope your school gives you access to the full text of these or similar papers: http://www.springerlink.com /content/w437x3j14w16r507/ , http://www3.interscience.wiley.com/journal/118511085/abstract? CRETRY=1&SRETRY=0 , http://jn.nutrition.org/cgi/reprint/70/2 /267.pdf)
If you use HPLC (high performance liquid chromatography), you could collect
the eluant from the column into different vials by time (say, change the
vial every 5 minutes), then test them all for antifungal activity. Note
that you will probably have to dry the fractions (evaporate the solvent)
and try to redissolve them in some other solvent (water if possible), since
many solvents themselves will kill fungi, so you don’t want false positives
coming from the solvent.
(1) Ahsan Sharif, Ejaz Ahmed, Abdul Malik, Naheed Riaz, Nighat Afza, Sarfraz Ahmad Nawaz, Muhammad Arshad, Muhammad Raza Shah and Muhammad Iqbal Choudhary. (2005) Lipoxygenase inhibitory constituents from Indigofera oblongifolia Archives of Pharmacal Research 28: (7) 761-764. (387.5 KB PDF)
(2) Esther Katuura, Paul Waako, John R. S. Tabuti, Remigius Bukenya-Ziraba, Jasper Ogwal-Okeng. (2007) Antiplasmodial activity of extracts of selected medicinal plants used by local communities in western Uganda for treatment of malaria African Journal of Ecology 45:(s3) 94 - 98.
(3) R.G. Coleman, G.M. Windrum, E.M. Hutton (1960) Studies on the toxicity
of Indigofera endecaphylla III. Separation of toxic
fractions from seed and herbage. Journal of Nutrition 70:267-271.
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