can anyone provide Best method for isolation of Peptides from ascidians?
Dear Velusamy Arumugam,
Herein the isolation method taken from a review article entitled "Bioactive peptides from marine sources: pharmacological properties and isolation procedures " by Abel Aneiros and Anoland Garateix published in Journal of Chromatography B, 803 (2004) 41–53:
Isolation methods of cyclic peptides
Purification procedures of these kind of cyclic peptides and depsipeptides isolated from sea animals like ascidians, sponges and mollusk, usually include initial extraction with methanol (MeOH), partitions of this extract with organic solvents of increasing polarities to render diverse organic fractions, and chromatographic steps on silica and Sephadex LH-20 columns, and the use of reversed phase C18 HPLC
for the final purification. As an example, a typical procedure of a bioassay guided
isolation applied to the obtainment of Onchidin B, a cyclodepsipeptide from the mollusk Onchidum sp. include an initial extraction from the freeze-dried animal with methanol. The methanol extract is concentrated in vacuum and the resulting viscous concentrate is partitioned between 10% aqueous methanol and hexane. The methanolic portion is then extracted with carbon tetrachloride (CCl4), and later
with dichloromethane (CH2Cl2). Each organic layer is concentrated in vacuum to yield the hexane, CCl4, and CH2Cl2 extracts. The CCl4 extract was submitted to flash silica gel column chromatography eluted with a gradient from dichloromethane to methanol resulting in five fractions. Fraction two was
further purified on a reversed phase C18 HPLC and resulted in purification of Onchidin B. Variants of this procedure additionally include for example steps of desalting of organic (alcoholic) fractions with the resin Amberlite XAD-2, and a separation step in a Sephadex LH-20 column eluted with methanol–water, just before the HPLC chromatography on the reversed phase C18 column.
To view the full publication, please use the following link:
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.327.608&rep=rep1&type=pdf
Hoping this will be helpful,
Rafik
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