Research Project: Biobased Pesticide Discovery and Product Optimization and Enhancement from Medicinal and Aromatic Crops

Location: Natural Products Utilization Research

2023 Annual Report

Objectives
1. Discover bioactive pesticides (fungicidal and herbicidal) that enhance quality and marketability of small fruits and specialty crops. 1.1. Discover novel biofungicides and bioherbicides from crude extract screening and bioassay-directed isolation approaches. 1.2. Isolation and identification of antifungal metabolites from actinomycete fermentation for control of important fungal plant pathogens. 1.3. Investigation of plant pathogenic fungi for phytotoxic and antifungal activities and synthetic modification of the isolated compounds to gain insights into structure-activity relationships. 2. Determine plant growth and post-harvest processing conditions to enhance or optimize commercially important bioactive compound concentrations. 2.1. Target medicinal and aromatic plant growth and post-harvest processing conditions to enhance or optimize commercially important bioactive compound concentrations.

Approach
The long-term objectives of this project are to identify, chemically characterize, and develop new weed and plant pathogen managing biochemical biopesticides from natural sources and enhance or optimize commercially important bioactive compound concentrations. Plant and microbial extracts will be sourced using a variety of approaches and methods as evidenced through previous publications by the authors. These extracts will be subjected to in-house bioassays which include both antifungal and herbicidal based assays as described below. A bioassay-guided investigative approach will be performed on bioactive extracts in an effort to identify the specific natural compound(s) responsible for the activity of the extract(s). In-house high-throughput bioassays will be utilized to identify bioactive constituents with antifungal and herbicidal activities. A second component of this project pertains to natural products obtained from medicinal and aromatic crops which are low-volume high-value commodities that have numerous applications in various industries such as food, beverage, food supplement, flavor and fragrance, perfumery and cosmetics, pharmaceutical, aromatherapy, and various consumer products. Sourcing these and other natural compounds in an economical manner directly from plants is constantly evolving which creates an opportunity for small and mid-size farms.

Progress Report
This is the third year of research for this project. This report also includes progress made as part of the NACA with the National Center for Natural Products Research (NCNPR) at the University of Mississippi in Oxford, Mississippi. This will also be the first year to report on progress made as part of the new NACA with Marshall University. For objective 1, discover bioactive pesticides (fungicidal and herbicidal) that enhance quality and marketability of small fruits and specialty crops, milestones have been met and significant progress has been made. Anthracnose is an economically important disease that affects strawberries globally and can lead to significant losses in both yield and fruit quality. The objective of this study is to identify EOs with antifungal properties against C. fragariae and other pathogenic fungi. We found 20 plant EOs has antifungal activity against anthracnose pathogens. Among these 20 EOs, the patchouli EOs (Pogostemon cablin) was further studied, and the major constituent’s patchouli alcohol exhibited antifungal activity against C. fragariae (isolate cf63) as well as other two pathogens C. gloesoporioides (one of the fungus that cause anthracnose in strawberries) and Botrytis cinerea that cause gray mold in strawberries. Either whole Patchouli EOs or its major constituent’s patchouli alcohol can be used to manage anthracnose disease in strawberries. Nicotine is a class of pyridine type natural products which was first found in tobacco. Nicotine has the effects of killing insects and regulating human physiological activities. Nicotine is also an important intermediate in the synthesis of the natural product niacin and niacinamide, which are essential vitamins for human. Nicotine acid derivatives can be found in many plant or microbial secondary metabolites. In order to discover lead compounds with high fungicidal or herbicidal activity, new niacinamide derivatives containing chiral flexible chains were designed and synthesized derived from the natural product niacinamide. The fungicidal and herbicidal activities of these compounds were tested. The fungicidal activity results demonstrated that the compound (S)-2-(2-chloronicotinamido)propyl-2-methylbenzoate exhibited good fungicidal activity (92.3% inhibition) against the plant pathogen Botryosphaeria berengriana at 50 µg/mL and the EC50 is 6.68±0.72 µg/mL, which is the same as the positive control (fluxapyroxad). This compound was not phytotoxic and could therefore be used as a fungicide on crops. Structure-activity relationships (SAR) were studied by molecular docking simulations with the succinate dehydrogenase of the fungal mitochondrial respiratory chain. Natural pesticides can provide an alternative approach for managing pests and diseases, as they often have different modes of action and lower toxicity to non-target organisms, such as beneficial insects, birds, and mammals. Yerba santa, meaning “holy weed” or “sacred herb” in Spanish, is a common name for several plant species in the Eriodictyon genus. The plants are native to western North America and their leaves have historically been used for centuries to treat various aliments among indigenous people of the region. Our studies on this plant species revealed that these plant species contain diverse groups of constituents, many of which have not been previously described. More than 10 novel compounds were isolated. Studies using germination and plant growth bioassays suggested that extracts and fractions of yerba santa possessed strong herbicidal activity against lettuce (Lactuca sativa), creeping bentgrass (Agrostis stolonifera), and ryegrass (Lolium multiflorum). These newly discovered activities from the extracts and fractions suggested the existence of desirable herbicidal compound(s) in yerba santa. In our previous work we have shown occurrence of antifungal and phytotoxic activities in the seeds of some Apiaceae plant family. We have investigated the antifungal constituents that can be used to control post-harvest decay of fruits and vegetables. Some of these constituents are essential oils and thus have high vapor pressure making them potential sources of fumigants. Most of the seeds we investigated have been used in culinary industry for long periods of time and ca be considered as non-toxic chemicals. We are making synthetic analogs of 4-isopropylbenzaldehyde, 4-isopropyl benzoic acid, 4-isopropylbenzyl alcohol, 2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enone, apiol and elemicin that have been isolated from the seeds of the plants in Apiaceae family. For objective 2, determine plant growth and post-harvest processing conditions to enhance or optimize commercially important bioactive compound concentrations, milestones have been met and significant progress has been made. The non-pungent analogs of capsaicinoids, were first identified in fruit of Capsicum annuum. Recent studies have reported that various biological activity characteristics of capsaicinoids are also characteristic of capsinoids albeit with less of the adverse effects associated with pungency. In support of a process NPURU patented last year, we investigated the effects of a C. annuum extracts and pure capsiate on metabolic disorder important receptors. The findings from this study suggest that capsiate will enhance glucose uptake effect and prevent lipid accumulation. Additionally, capsiate could inhibit the adverse effect of blood glucose lowering drugs belonging to thiazolidinediones class without compromising their main effects. This is the first report to reveal the multiple nuclear receptors agonistic action and glucose uptake enhancing property of capsiate from C. annuum along with its antiadipogenic effect indicating its potential in preventing the undesired adipogenic effects of full PPAR¿ agonists such as the glitazone class of antidiabetic drugs. In recent years, the popularity of copaiba oil-resin has increased worldwide due to its medicinal value and wide applications in industry. Despite its popularity, the oil has not been standardized by industry or regulatory agencies. Product adulteration in order to maximize profits has become a problem. To address these issues the current study describes the chemical and chemometric characterization of forty copaiba oil-resin samples by GC/MS. The commercial samples detected as outliers were further subjected to analysis using an SFC/MS method. Product adulteration with soybean oil was clearly detected, with each individual triglyceride in soybean oil being unambiguously identified. By combining these analytical techniques, the overall quality of copaiba oil can be assessed. University of Mississippi NACA - This year 1,081 plant samples were tested for biological activity in high-throughput assays. From the initial results, 9 samples were selected for advanced testing. To probe their immunomodulatory potential, 126 (including microalgae, kelp, mushrooms and plants) were evaluated in cell-based assays to detect activation/inhibition of molecular pathways in innate immune cells and redox homeostasis. Additionally, by implementing high throughput signal transduction metabolic panel assays, more than 10 pure phytochemicals were screened specifically for anticancer properties. Over 100 compounds were evaluated in a mechanistic assay designed to identify agents that modify the fungal cell wall. Hit compounds will be confirmed in beta- glucan unmasking assays, and advanced to in vivo studies for investigating their effects on the fungal microbiome in a mouse model of diabetes. The goal of this project is to identify natural products that can prevent microbiome-derived fungal infections associated with metabolic diseases. Mechanistic assays evaluating the inhibition of rate-limiting steps of the glycolytic pathway enzymes viz., hexokinase, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase, have been setup and over 10 lead compounds were screened for these enzyme(s) inhibitory potential. The goal of this project is to identify the mechanism responsible for the glycolytic inhibitory potential of these lead compounds and to use these enzymes as biomarkers in translational studies. Marshall University NACA - The initial approach of growing plants locally for study has been replaced by an approach of directly collecting or purchasing materials from qualified collectors of targeted plants/trees in West Virginia with appropriate collecting permits. It was felt that the agricultural aspect of the project should be a later phase, once positive results were obtained from plants extracts/purified compounds.

Accomplishments
1. Melissa officinalis essential oil as an effective potato sprout inhibitor. Potato (Solanum tuberosum L.) is the most important noncereal staple crop in the world and is the main contributor toward improving food security and reducing hunger. Control of sprouting is critical for potato storage since the sprouting leads to alterations in weight, texture, nutritional value, softening, shrinkage, and formation of toxic alkaloids. The primary methods for controlling sprouting of stored potatoes are by using sprout-inhibiting chemicals that are effective. Chlorpropham (CIPC) is the most efficient and cost effective; however, ARS researchers in Oxford, Mississippi, have shown that CIPC and its degradation products are hazardous for consumer health and the environment. The goal of this project was to identify plant essential oils (EOs) as sprout inhibitors or suppressors in potato (Solanum tuberosum L.). The results indicated that Melissa officinalis L. EO inhibited sprouting. The pure isolated compounds, together with the major compound in M. officinalis EO (citral), were tested for sprout suppression on three potato cultivars (Ranger Russet, Terra Rosa, and Dakota TrailBlazer), which revealed that beta-citronellol reduced the sprout length and the number of sprouts in all three cultivars, while citral and (+)-alpha-terpineol reduced the sprout length and the number of sprouts in Ranger Russet relative to the two controls in all three cultivars. This is the first report on the use of Melissa officinalis L. EO as a sprouting inhibitor. Melissa officinalis whole EO can be utilized for the development of a new commercial products for sprout control of potatoes. Such products would reduce toxic chemical residues in potatoes and contribute to improved human and environmental health.

2. Establishment of quality control standards for Eriodictyon sp. Eriodictyon species, commonly known as yerba santa, are plants native to the Southwestern United States and northern Mexico. The plants are known for their medicinal properties and have been used by indigenous people for centuries to treat various ailments, particularly respiratory conditions. Despite a long history of traditional use, a full chemical characterization of many of the species is lacking. In addition, the constituent range of these species has not been comprehensively reported. ARS researchers in Oxford, Mississippi, initiated this investigation at the request of the American Herbal Pharmacopoeia (AHP) to develop analytical methods for the chemical characterization and quantitative determination of the primary constituents in yerba santa (Eriodictyon spp.). Over 100 plant samples were collected by 18 scientists/botanists and two companies from California, Arizona, Oregon, and New Mexico to give a comprehensive data set. Our study describes the development and validation of an UHPLC/DAD/MS method for the investigation of 111 samples belonging to six different Eriodictyon species, viz., E. californicum, E. angustifolium, E. trichocalyx, E. crassifolium, E. tomentosum, E. traskiae, and E. capitatum. Fourteen compounds, comprised of flavonoids and phenolic acids, were quantified. To the best of our knowledge, this work encapsulates the most comprehensive data set currently available for the chemical characterization and quantification of the primary constituents in Eriodictyon species. This work has been reported as part of the “American Herbal Pharmacopoeia and Therapeutic Compendium-Yerba Santa Leaves & Tops Eriodictyon californicum and related species-Standards of Identity, Purity, Analysis, and Quality Control”.

Review Publications
Lee, J., Wang, M., Mondal, G., Khan, I.A., Yates, C.R. 2022. Development of a GC/Q-ToF method coupled with headspace solid-phase microextraction to evaluate the in vitro metabolism of ß-Caryophyllene. Molecules. https://doi.org/10.3390/molecules27217441.
Kumarihamy, M., Tripathi, S., Balachandran, P., Avula, B., Zhao, J., Wang, M., Bennett, M., Carr, M.A., Micheal, L., Ocean, W.I., Marquart, M.E., Nanayakkara, D., Muhammad, I. 2022. Synthesis and inhibitory activity of machaeridiol- based novel anti-MRSA and anti-VRE compounds and their profiling for cancer- related signaling pathways. Molecules. https://doi.org/10.3390/molecules27196604.
Thoma, J., Cantrell, C.L., Zheljazkov, V.D. 2022. Evaluation of essential oils as sprout suppressants for potato (Solanum tuberosum) at room temperature storage. Plants. https://doi.org/10.3390/plants11223055.
Thoma, J., Cantrell, C.L., Zheljazkov, V.D. 2022. Effects of essential oil fumigation on potato sprouting at room-temperature storage. Plants. https://doi.org/10.3390/plants11223109.
Lee, J., Wang, M., Zhao, J., Ali, Z., Hawwal, M., Khan, I. 2023. Chemical characterization and quality assessment of copaiba oil-resin using GS/MS and SFC/MS. Plants. https://doi.org/10.3390/plants12081619.
Semerdjieva, I., Zheljazkov, V.D., Dincheva, I., Piperkova, N., Maneva, V., Cantrell, C.L., Astatkie, T., Stoyanova, A., Ivanova, T. 2023. Essential oil composition of seven Bulgarian Hypericum species and its potential as biopesticide. Plants. https://doi.org/10.3390/plants12040923.
Min, L., Shen, Z., Bajsa Hirschel, J.N., Cantrell, C.L., Han, L., Hua, X., Liu, X., Duke, S.O. 2022. Synthesis, crystal structure, herbicidal activity and mode of action of new cyclopropane-1,1-dicarboxylic acid analogues. Pesticide Biochemistry and Physiology. https://doi.org/10.1016/j.pestbp.2022.105228.
Wei, Z., Wang, Q., Min, L., Bajsa Hirschel, J.N., Cantrell, C.L., Han, L., Tan, C., Weng, J., Liu, X., Duke, S.O. 2022. Synthesis and pesticidal activity of new niacinamide derivatives containing a flexible, chiral chain. Molecules. https://doi.org/10.3390/molecules28010047.
Zheljazkov, V.D., Micalizzi, G., Yilma, S., Cantrell, C.L., Reichley, A.C., Mondello, L., Semerdjieva, I., Radoukova, T. 2022. Melissa officinalis L. as a sprout suppressor in Solanum tuberosum L. and an alternative to synthetic pesticides. Journal of Agriculture and Food Chemistry. https://doi.org/10.1021/acs.jafc.2c05942.
Shi, H., Zhai, Z., Min, L., Han, L., Sun, N., Cantrell, C.L., Bajsa Hirschel, J.N., Duke, S.O., Liu, X. 2022. Synthesis and pesticidal activity of new 1,3,4-oxadiazole thioether compounds containing a trifluoro-methylpyrazoyl moiety. Research on Chemical Intermediates. https://doi.org/10.1007/s11164-022-04839-x.
Semerdjieva, I.B., Radoukova, T., Cantrell, C.L., Astatkie, T., Kacanlova, M., Borisova, D., Zheljazkov, V.D. 2022. Essential oil composition of Pinus heldreichii Christ., P. peuce Griseb., and P. mugo Turra as a function of hydrodistillation time and evaluation of its antimicrobial activity. Industrial Crops and Products. 187(Part B). Article 115484. https://doi.org/10.1016/j.indcrop.2022.115484.
Zhang, J., Agarwal, A., Feng, Q., Tripathi, S.K., Khan, I.A., Pugh, N.D. 2023. Identification of botanicals that unmask beta-glucan from the cell surface of an opportunistic fungal pathogen. Journal of Dietary Supplement. https://doi.org/10.1080/19390211.2023.2201355.
Chae, H., Dale, O., Mir, T.M., Ashfaq, M.K., Avula, B., Walker, L.A., Khan, I.A., Khan, S.I. 2023. Juniper berry (Juniperus communis L.) extract regulates obesity markers through modulating PPAR-alpha, PPAR-gamma, and LXR: in vitro and in vivo effects. Journal of Medicinal Food. https://doi.org/10.1089/jmf.2022.0146.
Sparks, T.C., Sparks, J.M., Duke, S.O. 2023. Natural product-based crop protection compounds - origins and future prospects. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.2c06938.
Husain, I., Dale, O.R., Martin, K., Gurley, B.J., Avula, B., Chittiboyina, A.G., Khan, I.A., Khan, S.I. 2022. Screening of medicinal plants for possible herb-drug interactions through modulating nuclear receptors, drug-metabolizing enzymes and transporters. Journal of Ethnopharmacology. https://doi.org/10.1016/j.jep.2022.115822.
Pandey, P., Kumarihamy, M., Ibrahim, M.A., Ilias, M., Doerksen, R.J. 2023. In vitro and in silico studies of neolignans from Magnolia Grandiflora L. seeds against human cannabinoid and opioid receptors. Molecules. https://doi.org/10.3390/molecules28031253.
Avula, B., Katragunta, K., Osman, A.G., Ali, Z., Adams, S., Chittiboyina, A.G., Khan, I.A. 2023. Advances in the chemistry, analysis, and adulteration of anthocyanin rich-berries and fruits: 2000-2022. Molecules. https://doi.org/10.3390/molecules28020560.
Cao, L., Wang, M., Zhao, J., Peng, L., Cheng, J., Qiu, S., Khan, I., Li, X. 2022. Comparative analysis of chemical profiles of Radix Astragali between ultrafine granular powder and traditional sliced mate-rials. Medicinal Plant Biology. https://doi.org/10.48130/MPB-2022-0004.
Meepagala, K.M., Tamang, P. 2023. Antifungal activity of Poncirus trifoliata roots against Colletotrichum species. Agricultural Sciences. https://doi.org/10.4236/as.2023.143022.
Drumond De Souza, L.M., Costa Barreto, D.L., Da Costa Coelho, L., Amancio Teixeira, E.A., Nicolau Goncalves, V., Muzetti Ribeiro, J.P., Gontijo Rabelo, N., De Oliveira Alves, S.E., Da Silva, L.M., Cantrell, C.L., Duke, S.O., Henrique Rosa, L. 2023. Fungal biosurfactants: applications in agriculture, medicine, industry and in environmental bioremediation processes. In: Marcelino, P.R.F., Da Silva, S.S., Lopez, A.O., editors. Biosurfactants and Sustainability: From Biorefineries Production to Versatile Applications. Hoboken, NJ:John Wiley & Sons Ltd. p 243-254.
Costa Barreto, D., De Carvalho, C.R., De Almeida Alves, T., Zani, C., Cantrell, C.L., Duke, S.O., Rosa, L. 2023. Influence of genetics on the secondary metabolism of fungi. Natural Secondary Metabolites – from Nature, Through Science, to Industry. https://doi.org/10.1007/978-3-031-18587-8_22.
Matundura, J.O., Midiwo, J.O., Yenesew, A., Omosa, L., Kumarihamy, M., Zhao, J., Wang, M., Tripathi, S., Khan, S., Masila, V.M., Nchiozem-Ngnitedem, V., Muhammad, I. 2022. Antiplasmodial and antimicrobial activities of ent-abietane diterpenoids from the roots of Suregada zanzibariensis. Natural Product Research. https://doi.org/10.1080/14786419.2022.2158463.
Wang, M., Zhao, J., Avula, B., Lee, J., Upton, R., Khan, I. 2023. Chemical characterization and quantitative determination of flavonoids and phenolic acids in yerba santa (Eriodictyon spp.) using UHPLC/DAD/Q-ToF. Journal of Pharmaceutical and Biomedical Analysis. https://doi.org/10.1016/j.jpba.2023.115570.