We have prepared extract by using warm methanol soxhlet extraction, then removed chlorophyll by using hexane and then used petroleum ether to remove fatty acids.
I do not recommend the use of DPPH to determine antioxidant activity because you are not including the substrate that you want to protect against oxidation.
Agree to Frankel. DPPH is still questionable. I have another doubt. If you are removing fatty mater then what about lipophilic antioxidant ? viz Carotene, Vit E.Plz look these articles
You may test the following protocol in your laboratory:
Prior to extraction using methanol, it is preferred to defat the plant material using hexane to avoid cross contamination from lipids. Later on, filter the methanolic extract of plant through activated charcoal to remove pigments such as chlorophyll. Subsequently, the antioxidant activity of defatted methanolic extract of plant material can be tested using different in-vtro assays such as DPPH, ABTS, ORAC. The antioxidant activity contributed by lipid soluble components extracted in hexane fraction during defatting can be checked using modified DPPH assay using iso-octane.
Agree with Rohit, remove nonpolar material using hexane and you can further partitioned with chloroform and 60 % MeOH in order to separate moderately polar and polar constituents in the extract. Then do the antioxidant assay for fractions separately.
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