Corrie Anderson, of Columba College, has been awarded a Gold CREST for her project "The Study of Horopito Looking at its Insecticidal Properties and Chemical Analysis"
Mentors: Marina Roxburgh and Dr Dave Warren,Otago University
Supporters: Dr Nigel Perry; Chemistry Department,Otago University
Supervising teacher: Dr.Murray Vickers
Gold CREST medal presented by: Professor Keith Hunter FRSNZ
Corrie's Executive Summary states:
"Horopito is a native New Zealand plant which has been used from the time of the arrival of the Maori and Early Settlers for medicinal purposes, for stomach ache and as a painkiller. Horopito contains an interesting compound located within the plant’s leaves known as polygodial. This compound has been shown to have anti-fungal qualities (witnessed while completing my Sliver CREST award investigating the anti-fungal properties of Horopito). My Gold Crest award investigation looks at expanding our knowledge of Horopito with the main focus on insecticidal properties.
Information research found from previous articles and papers that have studied Horopito have found that the plant contains polygodial and that it has anti-fungal properties. Horopito has been found to have insecticidal properties when tested against the Australian Carpet Beetle.
This investigation was based on a few New Zealand Horopito studies showing chemical analysis and insecticidal properties possibly caused by a compound known as a polygodial.
The aim of this study was to investigate Horopito by fractionating seasonal extracts and analyse the major fractions to explore any possible chemical and insecticidal properties towards aphids.
This study considered the chemical properties of Horopito over four seasons along with extracts which could potentially help horticulturalists with insect-infested plants. Aphids, being a common insect to infest plants, were seen as a visible marker regarding possible insecticidal affects from Horopito extracts.
The experiments were carried out in two stages:
1) The analysis of Horopito’s chemical structure
This was carried out with several techniques including thin layer chromatography (TLC), column chromatography, rotary evaporating, Nuclear Magnetic Resonance (NMR), and Infrared spectra (IR) which looked at seasonal variation over the course of a year while investigating compounds found within Horopito known as polygodial. Four extracts were taken of Horopito leaves i.e. Autumn, Winter, Spring and Summer. Each category had column chromatography undertaken on the leaf extracts which were divided into fractions of 12 which were then rotary evaporated. Through this process the method for column chromatography was constantly being refined for further accuracy in the results. Part of the project was to isolate polygodial into 1-2 fractions as it would be tested later on for insecticidal properties. This was achieved by dividing fractions 6 into 3 parts (A, B & C) and fraction 7 into 4 parts (A, B, C, & D). Using this method, the fractions, dissolved in petroleum ether and ethyl acetate, were evaporated to leave behind the extract.
TLC was then carried out with fractions 6A, B & C and 7A, B, C, D and 8 being compared to a sample of polygodial (provided by Dr Nigel Perry). This would show the amount of polygodial present within each fraction. Fractions 7C and 7D for winter, spring and summer showed the largest amount of polygodial.
Following this experiment NMR analysis was carried out on both a winter and spring extract 7C and 7D as well as an extract that had been left open to the air for 6 months compared with the other fraction. The results of 13C NMR spectroscopy confirms the isolation of polygodial compounds with the comparison of peaks above 100 ppm. 13C NMR of sample 7B in air showed a large number of new peaks in the region 100 – 200 ppm. This showed the sample degraded over the six months leading 1H NMR analysis which would determine if the aldehyde group was being converted to a carboxylic acid. No new peaks were found in the region 8-14 ppm (1H NMR), which indicated that the polygodial was not being converted to a carboxylic acid.
The NMR results show that between the similar peak position e.g. in fractions 7C and 7D for both winter and Spring different carbon environments have slightly different ratios in peak sizes. The comparing of Spring fractions 7C and 7D show that 7C on average has higher stretched peaks than 7D. This suggests that possibly different isomers or that two very closely related molecules are present, e.g. one could have an extra CH3 group were formed in 7C compared to 7D.
A sample of the polygodial fraction was left to air for 6 months at room temperature. NMR showed that it had changed considerably. No evidence of carboxylic acid groups was found which was unexpected. IR was carried out on summer fractions 3, 6, 7A, 7B, 7C, 7D, 8, 9, 10, 11, & 12 as they contained the most significant weight. Fraction 7C and 7D showed peaks that had the same bonds as a polygodial.
2) The testing of fraction containing concentrated amount of polygodial (PGD) for the investigation into the insecticidal properties of Horopito
The insects used for the testing of insecticidal properties were aphids being tested on summer extracts 7C, 7D and winter extract 7C. Each test was repeated three times and filmed for 2 hours with 15 aphids in the petri dish containing a leaf brushed with three layers of the fraction either side.
Three petri dishes were compared; (1) the dish with the fraction extract, (2) a control with a leaf with nothing on it and (3) another dish with ethyl acetate that had the same procedure as the petri dish with the leaf extract. Ethyl acetate was used as the solvent with the extraction having been dissolved at a 10% concentration. This was used to eliminate the variable of the aphids being affected by the ethyl acetate with a leaf brushed on with the ethyl acetate (15 aphids also used in the petri dish).
The results from these tests showed a continuing increase in the percentage of immobilised aphids over the 2 hour period where the summer fraction 7C had an average of 82% immobilisation between the three trials. Fraction 7D had an average of 96% of aphids immobilised. Winter fraction 7C had an average of 82%. These results conclude that fraction 7D has the highest rate for insecticidal properties. Summer Fraction 7D showed to have the biggest difference between aphids immobilised compared to the ethyl acetate dish. Looking at the comparison between the two dishes at the time of 120 minutes, the difference was 12 ± 1 using a 95 % confidence interval. Both fractions winter 7C and Summer 7C showed to have the difference between aphids immobilised compared to the ethyl acetate dishes. The comparison between the fractions and their ethyl acetate dishes at the time of 120 minutes both had a difference of 10 ± 1 using a 95 % confidence interval.
In conclusion, it was found that Horopito did contain insecticidal properties with the highest optimal level of immobilisation of aphids was summer fraction 7D with 96% while fraction 7C for both winter and summer had a immobilisation rate of 82%. The TLC fractions 6A, B & C and 7A, B, C, D and 8 that were compared to a sample of polygodial. showed the amount of polygodial present within each fraction. Fractions 7C and 7D for winter, spring and summer showed the largest amount of polygodial. The IR of Fraction 7C and 7D showed peaks that had the same bonds as a polygodial.
The results of 13C NMR spectroscopy confirms the isolation of polygodial compounds with the comparison of peaks above 100ppm. 13C NMR of sample 7B in air showed a large number of new peaks in the region 100 – 200 ppm showing the sample degraded over the six months leading 1H NMR analysis which would determine if the aldehyde group was being converted to a carboxylic acid.
Further work to extend this project could be done to compare Horopito by looking at concentrations of polygodial in different parts of the NZ. Looking at Horopito nationally could also expand on the research of the plants insecticidal properties. Another extension of the project in the field of insecticidal properties is to look at the effect on other insects. For future testing of Horopito’s polygodial insecticidal properties, the extract could be made into a spray and could be tested for effectiveness on some pot plants, or other common plants that are affected by insects that are classed as pest to plants. Investigating the storage of PGD so that it does not degrade. E.g. keep oxygen/air away from it; dissolve in alcohol or other solvents; keeping at low temperature – deep freeze. GCMS on the fractions with PGD to find how many compounds are present.
This study on Horopito’s chemical structure and insecticidal properties shows the polygodial fractions of the Horopito plant has the potential to be used in an insect repellent on leaves that aphids destroy as it is a natural product (and is used as a food additive showing no harmful effect to humans yet). As a native species to New Zealand that prolific growth, it could be readily harvested for commercial use."