Bioactive Chemicals of Importance in Endophyte-Infected Grasses
Babu, J. (2009). Bioactive Chemicals of Importance in Endophyte-Infected Grasses (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/2608
Permanent Research Commons link: http://hdl.handle.net/10289/2608
Janthitrems are believed to be involved in the observed sporadic cases of AR37-infected perennial ryegrass staggers. Investigations into the role of janthitrems inperennial ryegrass staggers are difficult as isolation of the compounds from theryegrass is hindered by the inherent instability of these compounds. Thereforeattempts were made to isolate janthitrems from an alternative source, allowingthese janthitrem analogues to be used as surrogates for endophyte producedjanthitrems.Analysis of a series of Penicillium janthinellum cultures revealed the presence ofjanthitrems in a number of strains, including janthitrem B, janthitrem C and twonovel janthitrem compounds. Detailed one- and two-dimensional NMR and massspectral techniques identified the two novel compounds as 11,12-epoxyjanthitrems B and C, which were subsequently given the trivial namesjanthitrems A and D, respectively. Janthitrems B and C were isolated andidentified by NMR and revisions of some previously reported chemical shiftassignments were proposed. In addition to the janthitrems, penitrems were alsoidentified in two strains of P. janthinellum.The isolated janthitrem B was utilised for the development of efficient extractionprocedures, and for the determination of ideal storage conditions for janthitremcompounds. A method for the extraction and isolation of janthitrem B from a P.janthinellum culture was developed and optimised to yield 6 mg of janthitrem Bfrom 900 mL of fungal culture in two days. Stability studies of janthitrem Bindicated the ideal storage condition which minimised degradation was dry at−80 C where only 7% sample loss was observed over 300 days.Bioactivity studies of janthitrems A and B found these compounds to betremorgenic to mice, with janthitrem A (an epoxyjanthitrem) inducing moresevere tremors than janthitrem B. Insect testing also showed that both janthitremsA and B displayed anti-insect activity to porina larvae. Since theepoxyjanthitrems, which are associated with AR37 endophyte-infected ryegrass,were also shown to be tremorgenic and to display anti-insect activity, the insectresistance and the sporadic cases of ryegrass staggers displayed by AR37 may berelated to the presence of epoxyjanthitrem compounds.LC-UV-MS analysis of janthitrems A-D, penitrems A-F, lolitrem B, paspalinine,paxilline and terpendole C found these indole-diterpenoids to be more sensitiveby analysis using an APCI source as opposed to an ESI source. APCI negativeion LC-UV-MS required source induced dissociation in combination withincreased collision energy to suppress an acetate adduct peak, sourced from theacetic acid buffer. Negative ion MS2 and MS3 data produced more informativefragments compared to the conventional positive ion MS2 and MS3 data. Theavailability of both positive and negative ion LC-UV-MS methodologies willallow future endophyte products to be more thoroughly screened for differentclasses of secondary metabolites.Extracts of mouldy walnuts were analysed for the presence of tremorgenicmycotoxins after a dog was found to exhibit symptoms characteristic oftremorgenic mycotoxicosis. LC-UV-MS analysis of the mouldy walnutsidentified the tremorgenic mycotoxins penitrems A-F, thus confirming theveterinarian's tentative diagnosis of canine tremorgenic mycotoxicosis the firstreported case in New Zealand.
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