Even after filtering and centrifugation, the extract was a complex cocktail of compounds. Further refinement was needed to isolate the active parts of the extract; liquid chromatography was used fractionate the samples.
The structure of the active agent was unknown; activity may be due to an AMP, as in shown on medfly eggs. 100,000xg centrifugation removed any large proteins,
peptides of 3kDa would remain in solution (Kuo et al. 1996; Marchini et al.1997).
The downstream effects of using protease enzymes were deemed risky with regards to bacteriological tests as the enzyme may remain active and attack bacterial
proteins. Liquid chromatography (LC) was the chosen method as it would separate any peptides from solution but also remove all non-active components of the extract leaving an active fraction. This method would also leave any AMP’s intact for further analysis (Selsted et al. 1993; Wright and Goodacre. 2012).
C18 SPE was used to separate molecules by polarity from the extract before advancing to HPLC, this was the best available method for isolating AMP’s. C18 method outlined by Conceição was utilised ddH2O was used in place of deionized water (Conceição et al.2006).
4.1.1. SPE C18 reverse phase chromatography extraction. Reverse phase C18 separation produces fractions using a hydrophobic stationary
As a mobile phase is run through on a gradient becoming less hydrophilic; this causes disassociation of molecules from the c18 medium and elute out. The mobile changes from 100% H2O (highly polar) to 100% acetonitrile (MeCN) a non-polar solvent, (Plattner et al. 1977).
C18 set up
The protocol was carried out in a fume hood to prevent any chemical vapours from escaping and to keep all hazardous waste contained. Male and female extracts had separate cartridges to prevent cross contamination.
Cartridges were conditioned with 100%methanol to activate the medium. The column was washed thoroughly with acetonitrile (80%) flushing out residual. The column was washed again with the lowest concentration of acetonitrile (5%), this conditioned the medium for the sample loading. Acetonitrile was diluted with sterile ddH2O.
0.1% trifluoroacetic acid (TFA) was used V/V to acidify the solution protonating the peptides for better separation as recommended by sigma Aldrich.
A plunger was used to provide 20N of downwards force to the cartridges to increase the flow rate. The mobile phase increased in 5% increment from 0% to 100% MeCN, each fraction was collected in a separate tube. 5% increments produced a finer range of separations, for better isolation of the active compound(s).
Reconditioning cartridges between runs and testing fractions. Cartridges were flushed multiple times with 100% methanol between each run clearing any residue ready for a fresh run. The protocol above was repeated for male and female extracts, 8X concentrated solutions were run through. Each C18 cartridge had a 1ml capacity so concentrated extract was used.
The fractions were diluted to 50% minimum withddH2O to ensure they froze at -80oC before freeze drying. Fractions were rehydrated to their original volume and plated onto E. coli plates. They were incubated at 27.5oC for 24 hours and observed for any plates with inhibition of growth this would indicate which fraction contained the antimicrobial and by extension the hydrophobicity of the molecule.
C18 cartridge cleaning and reconditioning
Cartridges were twice flushed with 100% methanol followed by a wash with 0.1% TFA/70% MeCN. 0.1% TFA/ddH2O was flushed through then rinsed with ddH2O and freeze dried, lyophilising all traces of acid, desalts the system ready to be reused.
4.1.2. Hilic solid phase extraction separation.
C18 did not give the desired degree of separation. It was hoped Hilic separation would prove more fruitful. The Hilic separation uses hydrophilic interactions binding to hydrophilic molecules. Separation protocol is like that of C18, instead of
decreasing the mobile phase polarity it increases using a water concentration gradient, (Wilson et al. 2007).
Hilic cartridges were set up identically to the C18, conditioned with 100% methanol. The samples were dissolved in minimal water and loaded and given sufficient time to bind to the medium, the run through (RT) was collected this contained all unbound molecules. 100% acetonitrile was then pass through the column eluting the most hydrophobic molecules. The concentration of acetonitrile was decreased in increments of 10% using ddH2O until finally 100% water was passed through the cartridge.
20N of positive pressure was applied, each fraction was also diluted with H2O to allow them to freeze. The fractions were tested against E. coli identically to the C18 fractions. Cartridges were reconditioned in an identical method to the C18 cartridges
The sample was initially dissolved in water, this had the undesired effect of not allowing the extract to bind to the medium instead it ran through the column. We attempted to dissolve the extract in acetonitrile however the extract did not dissolve until it was diluted to 20% with ddH2O, this was not optimal for separation.
Methanol was tried as it is more polar than acetonitrile the extract would not dissolve. Hilic separation was no more effective at fractionation than C18, however, the technique may be revisited as a potential refinement stage prior to introducing the sample to HPLC, by so doing removing much of the inactive agents from solution and reducing the overall “noise” of the profiles.
Figure 10: Separation gradient with regards to the mobile phase in Hilic and Reverse phase as well as the types of molecules which will elute out at a specific polarity (Heckendorf and Jonsson Copyright © 1995-2017. The Nest Group).