The process

Extraction

Drying (through a freeze dryer or air) and pulverising the mycelia as a first step increased the surface area, expedited the extraction process of metabolites using organic solvents like ethyl acetate or hexane. Oven-dried mycelia formed a solid surface which prevented solvent extraction. The presence of water on wet and intact mycelium prevented hydrophobic solvents from penetrating. MeOH and other alcoholic solvents which are miscible with water presumably rupture cell membranes and extract a greater amount of polar metabolites and endocellular materials (Colegate and Molyneux, 2007). They are, therefore, used to extract metabolites from wet mycelium directly. The downside of MeOH is it is not selectively extracting metabolites but adds to the extract impurities and then requires additional work of removing them. With dried material, ethyl acetate or low-polarity solvents will only rinse or leach the sample, providing to some extent a ‘cleaner’ crude extract (Colegate and Molyneux, 2007). Preliminary study showed no hydrophilic insecticidal metabolites were found from the mycelium. The extraction process in the future could just focus on using ethyl acetate (EA) from dried and pulverised mycelia.

Separation

The aim of the separation is to purify a substance by leaving behind impurities. Two main separation processes were used; (i) separation based on the polarity of the metabolites and (ii) the molecular size of the metabolites (Colegate and Molyneux, 2007). The first process immobilised all extracted metabolites on a substrate (silica gel) and gradually eluted the metabolites from the substrate according to their affinity to the different solvent systems. In this study, the insecticidal metabolites were mostly eluted from the mycelial extract using a solvent system of 70% hexane and 30% diethyl ether. These insecticidal metabolites were thus non-polar metabolites. The use of silica gel, however, has its downside. Silica gel-based supports frequently can irreversibly retain polycyclic ethers, brevetoxins and some cyclic peptides and cannot be desorbed even with polar solvents (Colegate and Molyneux, 2007). The use of a second process, differing from the first approach, relying on size exclusion was crucial. It helped to confirm that all insecticidal metabolites were eluted from LH20, and,

in this project, all metabolites of interest were eluted in the first few fractions collected. The other coloured metabolites, including the red metabolites, moved down the LH20 slowly and were not insecticidal. LH20 fractions showed beauvericins were abundant, eluted first, and bassianolide came in together with the beauvericins. There were no other obvious insecticidal metabolites. This study therefore recommends the use of LH20 resin to collect semi-pure insecticidal metabolites. Separation of semi-pure metabolites using size exclusion technique (LH20) was easier and faster than immobilising them on substrate (silica gel) and eluting using solvents of different polarity. Both techniques reduced impurities and provided concentrated amounts of insecticidal material for further purification.

Analytical and semi-preparative HPLC

Bioassays using insects, unlike microbes, require a higher amount of purified material to demonstrate bioactivities. Analytical fractionation on HPLC did not provide the required amount unless the column was overloaded with a concentrated semi-pure sample. Separation of peaks was still possible but with broad peaks. The purity could be compromised. Yet the fractions collected were sufficient to provide decent amounts for meaningful bioassays, pinpointing the exact insecticidal peak(s). The bioactivity was clearer as well when the collection were carried out 2-3 times and pooled.

The analytical column used in HPLC is not designed for major collections. A semi-preparative column was thus purchased to handle to the load of semi-pure samples separation and purification. Assisted by a fraction collector, pure metabolite(s) estimated at 300 mg from 1 ml fractions were able to provide toxicity assay on aphids. The rest of the purified material could be used for MS analysis.

HPLC gradient system

A HPLC gradient system was chosen to elute the metabolites gradually with the polar metabolites eluting first and the non-polar metabolites eluting as the column was saturated with more non-polar solvents. Trial and error showed the metabolites would only elute efficiently when the MeCN reached

95% at the 22nd _{min and kept at this concentration for 7 min. This system was modified to enable all}

the insecticidal non-polar metabolites to elute without overlapping. One recommendation however would be to prolong the 95% MeCN hold to 10 min to elute any insecticidal metabolite that were less polar than bassianolide (Figure 4.22). It could be there was another insecticidal metabolite which was not effectively separated using the current gradient system. This final peak was believed to contain a mixture of 'junk' material. The mixture of metabolites of this peak could have contributed to the high insecticidal activity observed but there is another possibility that it just needs some adjustment to the gradient system to elute another pure insecticidal metabolite.

Polarity

eluted on a RPC18 column. Polar metabolites were eluted first followed by nonpolar metabolites. The polarity of the insecticidal metabolites from the most polar to the least polar is as follows: beauvericin H1, (m/z) 802.5 (32% of aphid mortality), beauvericin (m/z) 784 (33%), beauvericin A, (m/z) 798 (16%), beauvericin B (m/z) 812 (4.2%), bassianolide (m/z) 909.6 (77%). General MS scan

The general MS scan from (m/z) 50 - 1400 showed bassianolide was the largest insecticidal metabolite from the semi-pure mycelial extract. No oosporein (m/z) 306 (Ross et al., 1989) was selected for observation partly because it was never encountered in insecticidal fractions or reported as having insecticidal activity on its own. In addition, oosporein is normally detected in negative mode like organic acids.