Location: Natural Products Utilization Research
2015 Annual Report
Objectives
Objective 1: Discover new insecticidal active ingredients that are based on natural products and that are safe with respect to occupational exposure and with respect to the environment, including non-target effects.
Subobjective 1.1: Discover novel arthropod repelling biopesticides from mosquito repelling folk remedies.
Subobjective 1.2: Discovery of fungal compounds and natural product analogs with activity against permethrin-resistant and susceptible mosquitoes.
Subobjective 1.3: Discover novel bioinsecticide active ingredients from crude plant and fungal extract screening and bioassay-directed fractionation.
Objective 2: Participate in development of new products based on green biopesticides.
Subobjective 2.1: Develop and optimize fatty acids as natural topical and spatial arthropod repellents.
Subobjective 2.2: Development and optimization of the chromene derivative 131-1 as a biopesticide.
Approach
A bioassay-directed investigative approach will be performed on bioactive extracts in efforts to discover bioinsecticides and repellents for use against arthropod pests. General procedures for isolation of active compounds from plants and microbes will be utilized. Solvent extractions, liquid-liquid partitioning, column chromatography and thin layer chromatography will be employed as needed. Extracts, fractions and pure compounds will be tested for insecticidal and insect repellent activity in assays being carried out routinely at collaborator's laboratories. Identification of active compounds will be done using spectroscopic methods including mass spectroscopy (EI, CI, and high resolution ESI), Nuclear magnetic resonance (one- and two-dimensional). Chemical modification will be performed on compound(s) identified as "highly active" to improve activity or bioavailability.
Progress Report
This project was approved by OSQR and became effective mid-year on April 1, 2015. Its preceding temporary project terminated on March 31, 2015. Results from both projects will be reported here in whole and 12 month milestones will be used.
Larvicidal Thiophenes from Echinops sp.: Six thiophenes were isolated from the roots of Echinops transiliensis. Structure-activity relationships of these compounds together with those previously isolated were evaluated for their larvicidal activity against Aedes (Ae.) aegypti. Terthiophene was most active (LC50, 0.61 ug/mL). Ae. aegypti larvicidal activity of thiophenes increase with increase in the number of thiophene rings and the most important active site in the structure of thiophenes could be the tetrahydrothiophene moiety. In bithiophenes, Ae. aegypti larvicidal activity increased with increasing the number of acetoxy groups attached to C-3' or C-4' indicating that acetoxy groups may play important role in the larvicidal activity. Also, Ae. aegypti larvicidal activity of thiophene had a significant negative correlation with lipophilicity (LogP; r = -0.726*).
Larvicidal Constituents from Streptomyces sp. (caat52): EMBRAPA visiting fellow arrived at NPURU with a number of crude plant and bacterial extracts that were evaluated for activity in Ae aegypti larvicidial screens. Plants and bacteria are from collections made in the “Caatinga” region of Brazil with the region characterized by semi-arid and desert-like conditions. A methylene chloride extract of the growth medium from Streptomyces sp. (caat52) has produced the most active larvicidal extract screened at present. Partitioning and fractionation of this extract has produced a fraction with 100% inhibition at <4 ppm against Ae aegypti larvae. Structure determination of the bioactive constituents present in this fraction is underway.
Fatty Acid Development: Previous investigations of the American beautyberry, breadfruit inflorescences and Jatropha sp. oil by ARS scientists clearly demonstrated the effectiveness of evaluating folk remedies for lead compound discovery. The later two folk remedies led researchers to focus development on fatty acids. Specifically, the investigation that began in 2011 on structure-activity relationships (SAR) of mosquito repellent fatty acids is complete. The research done in 2011 through 2013 resulted in multiple manuscripts on the optimum fatty acids that should be targeted for spatial repellent development. The fatty acids with the highest level of repellency were decanoic acid (C-10:0), undecanoic acid (C11:0), and dodecanoic acid (C12:0). Fatty acid candles produced and developed with C-12 saturated fatty acids were examined using spatial repellent field bioassays in 2014. Such field traps had not been previously utilized by our group and we therefore have little useful data to report from 2014 spatial assay. Overall the spatial assays were inconclusive and conclusions could not be drawn from results obtained. Blends of essential oil constituents that may enhance the activity of the fatty acids alone have been chosen and developed into prototype candles for further evaluation. Exact compositions will not be disclosed here as they are still under evaluation and not protected by intellectual property.
Sweetgrass Investigation: A folk remedy, sweetgrass (Hierochloe odorata), has been used by Canadian native Americans to repel insects. ARS scientists completed sourcing of the plant material (Univ. Guelph colleagues), crude extract extraction, and K&D preliminary screening in 2014 and 2015. Preliminary screening indicated strong repellency data and a bioassay-directed fractionation was used to isolate the mosquito repelling constituents. Briefly, fresh oil was used for fractionation studies providing 12 fractions.
Three of these fractions were identified as having activity equivalent to DEET and were the subject of further fractionation and identification studies.
Chromene analogs: Twelve Chromene and chroman derivatives were synthesized and some of these analogs showed better mosquito repellent activity than 131-1 but with shorter duration of activity (3 days vs 2 days). Four chromene derivatives have shown larvicide activity against permethrin resistant Ae. egypti mosquito strain and adult mosquitocide activity.
Fungal compounds and natural product analogs against permethrin-resistant and susceptible mosquitoes: Three fungi have been identified to possess larvicide activity and one fungi to poses adult mosquito activity. A lactone was isolated from one of the fungi and analogs of this constituent were synthesized. One analog was identified to possess better mosquito adulticide activity. The naturally occurring lactone also showed larvicide activity against permethrin resistant mosquito larvae.
Ammi visnagin investigation: From the seeds of Ammi visnagin, two moderately active constituents against mosquito larvae were isolated and identified.
Accomplishments
1. Several natural compounds from Echinops plant species serve as potent mosquito larvicides. Mosquitoes transmit pathogens that cause serious human diseases including malaria, Japanese encephalitis, yellow fever, dengue and filariasis. Insecticides from various chemical groups are the basic tools used for management of mosquito populations. Due to continuous use of insecticides, mosquitoes have developed resistance against these chemicals and vector population management has become difficult. Natural product-based products have gained special importance as potential new pesticides. In the continuing study of the natural compounds from the globe thistle genus (Echinops sp.), ARS scientists with the Natural Products Utilization Research Unit in Oxford, MS isolated six known natural compounds from Echinops transiliensis. Researchers describe for the first time the isolation and structure elucidation of these compounds and the relationship between the structure and the activity of these natural compounds as larvicides against the yellow fever mosquito, Aedes aegypti. Studies such as these provide a starting point for utilizing the natural compound (thiophene) structure scaffold as a synthon for developing insecticides. Utilization of one of the isolated compounds, terthiophene, directly as a bioinsecticide for further studies is warranted and is being pursued.
Review Publications
Nakano, H., Ali, A., Rehman, J., Mamonov, L., Cantrell, C.L., Khan, I.A. 2014. Toxicity of thiophenes from echinops transiliensis (Asteraceae) against aedes aegypti (Diptera: Culicidae) larvae. Chemistry and Biodiversity. 11(7):1001-1009.
Godinho, V.M., Goncalves, V.N., Santiago, I.F., Figueredo, H.M., Vitoreli, G.A., Schaefer, C.E., Barbosa, E.C., Oliveira, J.G., Alves, T.M., Zani, C.L., Policarpo Jr., A., Murta, S.M., Romanha, A.J., Kroon, E., Cantrell, C.L., Wedge, D.E., Duke, S.O., Ali, A., Rosa, C.A., Rosa, L.H. 2015. Diversity and bioprospection of fungal community present in oligotrophic soil of continental Antarctica. Extremophiles. 19:585-596.
