Accumulation of sesquiterpene lactones in pyrethrum extract
Creators: name: Dar, NG
Publisher: name: University of Tasmania
Year: 2023

description: Pyrethrum oil sourced from Tanacetum cinerariifolium is the main commercial source of the natural pesticide, pyrethrum oil with the active components being pyrethrin esters. Pyrethrum oil also contains sesquiterpene lactones (STLs) with pyrethrosin being the major STL. Following the refining process, pyrethrins are diluted from 80% to 53% with paraffinic naphthenic (LPA) carrier oil and pyrethrosin, which makes up 0.2-0.4% of the final product, is less soluble in the diluted oil and precipitates during storage. These crystals have been reported to block the nozzles during insecticide application. In addition, it has been reported that exposure of humans to pyrethrosin elicits an allergenic response. The solubility of pyrethrosin in pyrethrin oil diluted with a carrier oil is a major factor that compromises the quality of pyrethrin oil.This study focuses on two aspects: pyrethrosin solubility in stored pyrethrin oil in the presence of carrier oils and the investigation of the biosynthetic pathway for the production of STLs. Firstly, pyrethrum STLs were characterised and quantified using different techniques, including X-ray crystallography, polarised light microscopy and nuclear magnetic resonance (NMR). The precipitate was found to be pyrethrosin, despite the tentative identification of seven other STLs in the oil including cyclo-pyrethrosin, β-cyclo-pyrethrosin, dihydro-β-cyclopyrethrosin, C7-C8 guaianolides, tatridin A and dihydro-tatridin A and C7-C8 eudesmanolide, chrysanin. A method for quality control prior to distribution of the commercial product using polarised micoscopy was developed.Quantitative analytical methods developed in this study to analyse pyrethrum oil firstly trialled the use of liquid chromatography, as the pyrethrin esters are thermally labile however, coupling this with detection using Mass Spectrometry (MS) with Electrospray Ionisation (ESI) was non quantitative as the esters were found to degrade in the electrospray ionisation (ESI) chamber at high temperatures (400°C), whilst pyrethrosin condensed at lower temperatures (120°C, 200°C). Photo Diode Array (PDA) detection was also unsuitable for the simultaneous analyses of pyrethrosin and pyrethrin esters as co-eluting peaks required the specificity of Single Ion Monitoring (SIM). Similarly, when analysed by Gas Chromatography with detection by Flame Ionisation Detection (GC/FID), degraded pyrethrin I coeluted with pyrethrosin, resulting in an elevated baseline on the GC chromatogram. An optimal temperature gradient for GC was established which minimised the degradation of pyrethrin esters allowing for the quantification of pyrethrosin in the trials undertaken in this study.With a view to selectively remove pyrethrosin from pyrethrum oil prior to distribution, the application of silica and octadecane columns were trialled. Silica was found to effectively retain 94% solubilised pyrethrosin in reverse phase chromatography using hexane as the mobile phase while normal phase chromatography using octadecane as the matrix was less effective. Centrifugation was a superior method for removing precipitated pyrethrosin crystals when compared to filtration (0.45μm).The precipitation of pyrethrosin from oil is facilitated in the industry by storage of refined oil, diluted with carrier oil at -10°C, for a period of months, incurring significant costs to commercial operations. This study established the rate of precipitation under different storage conditions in several time series trials. The agitation of oil during storage to provide nucleation sites only slightly increased the rate of crystallisation. The study showed that the majority of the crystals were formed within the first 10 days at -10°C. Subsequent trials confirmed that the rate of precipitation was independent of storage temperature and predominantly occurred within the first 5 to 10 days of storage at both -10°C and 4°C. Larger-scale storage trials of pyrethrin oil at -10°C, 4°C, and room temperature showed that no significant difference in the rate of crystal formation was found for up to 90 days between the treatments.The blockage of spray nozzles by crystals could be alleviated by keeping pyrethrosin in solution. As such, the solubility of pyrethrosin in pyrethrin oil that had been diluted with different carrier oils was established. The solubility of pyrethrosin in mineral oil was found to be 9.4, 6.6, 5.93, 5.27, 5.1, 4.4 and 3.4 mg/g in 100, 90, 85, 80, 73, 70 and 55% diluted oil, respectively. A faster rate of precipitation was recorded for increasing amounts of carrier oil. The use of Isopar-M oil, mineral oil, and canola oil as alternative carrier oils were trialled at dilutions of 100, 80, 70, 55, 45, and 38 % relative to pyrethrum oil. All of the oils formed a partition except canola oil. Pyrethrosin concentration remained constant over time in all of the different dilution ratios of canola oil in pyrethrum oil.The localisation of STLs in the trichomes of pyrethrum flowers was confirmed by the selective extraction of the lactones by dipping the complete pyrethrum flower head in DCM. Only trace levels of esters were co-extracted, with the exception of cinerin 1. Environmental Scanning Electron Microscope (ESEM) images confirmed that the trichomes were emptied of oil without damaging the epidermis.An alternative to the removal of pyrethrosin from pyrethrum oil was to selective breed out the genetics for the production of STLs. Two seed lines named ‘Evil’ and ‘Virtuous’ had been selectively bred to produce high and low levels of pyrethrosin, respectively. Examples of each were propagated, vernalised and the flowers screened. The lowest level of pyrethrosin was 0.06%, detected in Virtuous, which was 24 times less than that recorded for Evil at 1.42%. Peaks tentatively identified as cyclopyrethrosin and dihydro-B-cyclopyrethrosin, on average, were more concentrated in Virtuous plants.ESEM was used to determine the size, density, and distribution of trichomes in both seed lines. On average, there was no significant difference in the trichomes number, although the spatial distance between organelles was found to be significantly different between the two seedlines.The genetic difference between Evil and Virtuous presented the opportunity to investigate the biosynthetic pathway of STLs. Three enzymes, TcGAS, TcGAO, and TcCOS, previously identified as providing the precursors to STLs, were compared by analysing the Gene Expression Ratio (GER) of these enzymes.The entire methodology of GER analysis was optimised for Tasmanian pyrethrum seedlines. Primers to allow for DNA amplification by Polymerase Chain Reaction (PCR) were designed along with the primers for two reference genes TcGAPDH and TcActin7, using a nucleotide sequence database GenBank of National Centre for Biotechnology Information (NCBI), and a public database for primer design Primer 3 of Free Software Foundation USA. The PCR program was optimised for primers specificity for all five genes. The amplified DNA of TcGAS, TcGAO, and TcGAPDH showed a single band on the gel with the expected number of base pairs (bp) when referenced to a ‘ladder’ of known gene fragment sizes, however, a double band was observed for TcCOS, and this may have been due to the formation of primer dimers.Real-time quantitative PCR (RT-qPCR) was used to establish a DNA standard curve with good linear regression efficiency of TcGAS (efficiency:1.00, R<sup>2</sup>=0.9995), TcGAO (efficiency:0.97, R<sup>2</sup>=0.9996), TcGAPDH (efficiency:1.01, R<sup>2</sup>=0.9997), and TcCOS (efficience:0.99, R<sup>2</sup>=0.9994). Melt curves analysis confirmed the purity of DNA for all the genes except for TcCOS, which showed a double peak.Each gene was sequenced, and the BLAST database showed the TcGAPDH sequence matched 93% with the related species Helianthus annuus. The sequences for TcGAS, TcGAO, and TcCOS matched 100%, 97%, and 100% respectively with Tanacetum cinerariifolium and TcActin7 matched 96% with Chrysanthemum morifolium ...The relative rate of expression for the three genes in Evil and Virtuous was compared using the technique known as GER analysis. The RNA of six Biological Replicates (BRPS) from stage 3 flowers of each seedline were extracted. The RNA Integrity Number (RIN) for each BRP was &gt;7, confirming the quality of the RNA. The Reverse Transcriptase qPCR (RT-qPCR) of TcGAPDH showed good Cq differences for both seedlines but the melt curve of the cDNA from the Evil seedline presented two peaks rather than a single DNA amplicon. Sequence analysis of the TcGAPDH revealed that the two peaks of the amplicon were due to nucleotide degeneracy of the sequence in Evil BRPS. In GER analysis by RT-qPCR, the cDNA segments of the 12 BRPS were amplified, and the expression ratio of each gene was calculated against two reference genes using the Relative Expression Software Tools (REST) method. The results showed TcGAS expression is down-regulated (p=0.025), while TcGAO (p=0.59) and TcCOS (p=0.14) were at the same level in Virtuous relative to Evil plants. The down-regulated expression of TcGAS correlated with lower pyrethrosin concentrations but did not correlate with the overall increased levels of other STLs, which were higher in Virtuous compared to Evil.Down-regulation of TcGAS, and the implied decrease in the biosynthesis of germacrene A, a precursor to STLs early in the biosynthetic pathway, may provide for the redirection of resources to other secondary metabolites in Virtuous plants such as flavonoids or other minor bioactives.This study provides knowledge to reconsider existing processes for the production of quality pyrethrum oil in terms of pyrethrosin solubility and storage.