Location: Natural Products Utilization Research
2018 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 is the third full year of research for this project since it was newly created. For Objective 1, discovery of new insecticidal active ingredients that are based on natural products, milestones have been met and significant progress has been made.
The culture filtrate of a plant pathogenic fungus that infects English ivy (Hedera helix L., Araliaceae) was investigated for mosquitocidal constituents by bioassay-guided isolation. The fungus responsible for pathogenic effects on the plant H. helix has been identified as Diaporthe eres Nitschke by molecular techniques. The mosquito adulticidal constituent in the culture filtrate was identified as 3,4-dihydro-8-hydroxy-3,5-dimethylisocoumarin (1) by spectroscopic techniques. Laboratory bioassays showed that (1) had larvicidal activity against permethrin-susceptible and -resistant Aedes aegypti strains. This compound was not active as an adulticide when tested by topical bioassay. Several analogs of (1) were synthesized and had better mosquitocidal activities than the naturally occurring (1) constituent.
One of the major compounds from cashew nut shell liquid that showed mosquito adulticide activity was modified via synthetic methods to yield mono and di-methylated analogs. Three more larvicidal compounds were also isolated. These compounds will be tested in the bioassays to get larval and adult LC50 values in USDA-ARS in Gainesville laboratory.
A systematic bioassay-guided fractionation of methylene chloride extracts of the aerial part of Artemisia arborescens was performed in order to identify its phytotoxic and larvicidal compounds. Two lignans were isolated, sesamin and ashantin, that inhibited growth of Agrostis stolonifera (bentgrass), a monocot, and Lactuca sativa (lettuce), a dicot, at 1 mg mL-1. In a dose-response screening of these lignans for growth inhibition against Lemna paucicostata (duckweed), ashantin was the most active with an IC50 of ca. 224 µM. The mode of action of these compounds is still unknown. In mosquito larvicidal bioassays against Aedes aegypti, isolated pure compounds sesamin and ashantin did not show any activity at the highest dose of 125 mg/L against 1-d-old Aedes aegypti larvae. In bioautography bioassays for antifungal activity using Botritis cinerea, Fusarium oxysporum, Colletotrichum fragariae, Colletotrichum acutatum, and Colletotrichum gloeosporioides, ashantin and sesamin were inactive at the highest amount tested, 5 micrograms.
Malabar spinach, Basella alba, is a popular green leafy vegetable in the Basellaceae family native to tropical Asia. From an infected leaf showing necrosis a colony of three fungi were cultured in potato dextrose agar (PDA) plates and identified via molecular techniques. One of these fungi was identified as a member of Phoma genus. The identity of this fungus was determined as Phoma pomorum. The culture filtrates were extracted with ethyl acetate, dried over anhydrous Na2SO4 and evaporated to obtain brown viscous liquids. The ethyl acetate extracts were larvicidal. In order to isolate active compounds, more ethyl acetate extract is needed. We are in the process of growing large scale (60 L) of fungal broth in potato dextrose broth (PDB). Two growth media, PDB and Czapek-Dox (CZ) were tested for the production of metabolites. Chromatographic analysis of the ethyl acetate extracts of the culture filtrate showed that PDB was a better medium to generate more metabolites. Isolated constituents will be investigated for mosquitocidal activity.
Thirty-seven phytophenolics and their synthetic analogues were evaluated for activity against two protozoal pathogens, Leishmania donovani and Plasmodium falciparum (D6 and W2 clones), respectively. 4,6-Dimethoxyaurone demonstrated the highest activity with IC50 values of 13.2 and 16.9 lM against L. donovani and P. falciparum (W2 clone), respectively, without undesired cytotoxicity against VERO cells. The moiety having two benzene rings was critical to maintain the antiprotozoal activities based on the observation that both coumaranones and chromones were inactive while other test compounds, including coumarans and aurones, remained active. There was no correlation between antiprotozoal activities and previously evaluated insect antifeedant activity against common cutworms (Spodoptera litura). Flavonoids, including aurones, pterocarpans, and lignan like dihydrobenzofurans, structural analogues of coumarans, are abundant in fruits and vegetables, so these phytophenolics may act as natural antiprotozoal agents in humans.
For Objective 2, participate in development of new products based on green biopesticides, milestones have been met and significant progress has been made.
Undecanoic acid (C-11) and dodecanoic acid (C-12) have been incorporated into a new delivery device by a commercial collaborator and the device is being evaluated as a spatial repellent. Last year’s efforts to include both C-11 and C-12 in the delivery device which uses a pad and heat were not successful. The fatty acids evaporated too quickly to be useful in such a device. This new device applies a lower temperature, which should aid in the longer delivery of the fatty acids and hence a better duration.
Related to the above technology, fatty acid methyl esters of C-11 and C-12 have been synthesized in an effort to both improve the mosquito repellency and improve the duration of activity of the natural C-11 and C-12 fatty acids. The optimum chain length for the esters has been determined and a highly active analog has been produced and shown to be more active than DEET in the K&D module bioassay system. Duration studies will be performed on these and other analogs produced synthetically. In-vivo evaluations of the most active compounds against Ae aegypti will be performed following the large scale synthesis of the analogs.
Large scale synthesis of 131-1 has been completed and a CRADA has been signed with a private sector partner for the development of 131-1 into a commercial product. Additional arthropod tested will be completed by the commercial partner as part of the CRADA agreement.
Accomplishments
Review Publications
Meepagala, K.M., Estep, A.S., Clausen, B.M., Becnel, J.J. 2018. Mosquitocidal activity of a naturally occurring isochroman and synthetic analogs from the plant pathogenic fungus, Diaporthe eres against Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology. 55(4):969–974. doi:10.1093/jme/tjy016.
Labruzzo, A., Cantrell, C.L., Carrubba, A., Ali, A., Wedge, D.E., Duke, S.O. 2018. Phytotoxic Lignans from Artemisia arborescens. Natural Product Communications. 13(3):237-240.
Morimoto, M., Cantrell, C.L., Khan, S., Tekwani, B.L., Duke, S.O. 2017. Antimalarial and antileishmanial activities of phytophenolics and their synthetic analogues. Chemistry and Biodiversity. DOI: 10.1002/cbdv.201700324.
