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

Location: Natural Products Utilization Research

2022 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 second year of research for this project. 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. 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. The genus Salvia includes around 900 species distributed throughout the world and is the largest genus in the economically and medicinally important plants of the Lamiaceae family. The secondary compounds of Salvia species have been extensively studied for numerous biological activities. The genus Salvia contains several allelopathic species and extracts from three of these species, Salvia miltiorrhiza, S. hispanica and S. columbariae, were tested in this study for phytotoxicity. Although there are several reports of phytotoxins from Salvia, there are no reports of phytotoxins against higher plants from Salvia miltiorrhiza. The abietane diterpenes tanshinone I and tanshinone IIA were identified as the compounds responsible for phytotoxicity. Natural products are a promising source for the development of new pesticides with alternative mechanisms of action. Sesquiterpene lactones (SLs) are one of the largest families of natural products and a broad range of bioactivities have already been reported. SLs are mainly isolated from the aerial parts of the family of the Compositae although they can also be found in other plant families, such as Umbelliferae, Lauraceae, and Magnoliaceae. Recent isolation studies have led to the discovery of a novel family of sesquiterpene lactones commonly named C17-sesquiterpenoids. In this study, we evaluated the phytotoxic and antifungal activity of a novel family of natural C17-sesquiterpenoids and examined the relationship between chemical structure and activity. Many tested compounds exhibited a strong, phytotoxic activity. Lappalone and pertyolide B were the most potent molecules from the tested group. Regarding the antifungal activity, pertyolide B presented significant activity against Colletotrichum fragareae and Fusarium oxysporum. Pesticides and fertilizers play crucial roles in modern agriculture. Due to the rapid evolution and spread of pesticide resistance, discovery and development of new pesticides with new structures and/or mechanisms of actions is needed. Heterocycles with nitrogen atoms are important structural units in many pesticides. Among them, 1,2,4-triazole is an important five-membered nitrogen-containing heterocycle which has led to the discovery of many agricultural fungicides used for crop protection such as propiconazole, tebuconazole, propiconazole, and cyproconazole. In a study in collaboration with colleagues in China, a series of 1,2,4-triazole derivatives containing dioxolane ring motif were designed and synthesized. Some of the synthetic 1,2,4-triazole derivatives exhibited good fungicidal and plant growth regulation activity. These new fungicides are structurally different from existing fungicides that act through inhibition of ergosterol synthesis. The antifungal activity of bacteria Psuedomonas vancouverensis isolated from a Farkleberry (Vaccinium arboreum) is being investigated. The bacterium Pseudomonas showed antifungal activity against the plant pathogenic fungi Colletotrichum fragariae, Botrytis cinerea, and Phomopsis obscurans on the PDA plate assay. The crude extracts also showed antifungal activity against C. fragariae in bioautography assay and now fractionation of the crude compound is under the way. Similarly, the antifungal constituents from a fungus Westerdykella multispora extracted from Sardis Lake are being investigated. The crude extracts from this fungus showed antifungal activity against C. fragariae in a bioautography assay and further fractionation and testing of the compounds responsible for the activity is underway. Approximately 176 crude extracts samples from NCPNR have been subjected to bioautography assay and six of them (Rhamnus beautifolia, Prunus pensylvanica, Senna obtusifolia, Eupatorium purpureum, Chamaecrista fasciculata, and Amphiachyris dracunculoides) showed antifungal activity against C. fragariae. Further fractionation and isolation process will be done. This is collaborative work with NCPNR, U. of Mississippi. 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. Finalizing a collaboration with Alabama A&M University, and Samford University. The overall goal of this project is to understand how Ocimum extracts modulate the biological actions of insulin and the molecular mechanisms of insulin resistance as they relate to the pathophysiology of diabetes while determining the bioactive compound(s) associated with antihyperglycemic activity. Sixteen Ocimum accessions consisting of five different species, viz. O. africanum, O. americanum, O. basilicum, O. campechianum, O. gratissimum, and O. tenuiflorum were cultivated by Alabama A&M University. An UHPLC/DAD/QToF method was developed for the quantitative analysis of 14 bioactive compounds present in Ocimum species. Meanwhile, methanol extracts of the 16 plant species were evaluated for a-glucosidase inhibitory activities. Several samples demonstrated comparable IC50 inhibitory concentrations as acarbose, a widely-prescribed anti-diabetic medication. A collaborative project between the USDA, ARS, NPURU and Alabama A&M University to develop high curcumin turmeric (Curcuma spp.) varieties for high tunnel production to sustain small farms in Alabama is underway. The project is funded by National Institute of Food and Agriculture, USDA. The goal for this project is to evaluate the growth, development and yield, and curcumin levels in response to techniques and production conditions that are known to potentially increase curcumin accumulation. An UHPLC/UV/MS method has been developed for the quantification of bisdemethoxycurcumin, demethoxycurcumin, curcumin, and aR-tumerone. About 400 samples collected in 2018-2020 have been analyzed, and samples collected in 2021-2022 are under investigation. Chemical Analysis of Eriodictyon Species: Method Development and Validation. This investigation was initiated at the request of American Herbal Pharmacopoeia (AHP) to develop and validate a reliable and efficient analytical method for the quantitative analysis of major constudents in different Eriodictyon species. In a separate focus on Eriodictyon, the antifungal constituents are being investigated. Eriodictyon crassifolium demonstrated antifungal activity against C. fragariae. B4. Peppermint and Copaiba Essential Oil Analysis. These are collaborative projects between the USDA, ARS, NPURU and NCNPR, University of Mississippi. The goal for these two projects is to develop simple. economical analytical methods which will allow anyone in the peppermint oil or copaiba supply chain to quickly and cheaply evaluate the authenticity and quality of the oils.

Accomplishments
1. Process for producing capsinoids from Capsicum sp.. Capsinoids, the non-pungent analogs of capsaicinoids (e.g. capsaicin), were first identified in fruit of the sweet pepper (Capsicum annuum) cultivar CH-19. Numerous subsequent studies have reported that many (but not all) of the types and degrees of biological activity characteristic of capaicinoids are also characteristic of capsinoids. The fruit of high-capsiate genotypes represent a commercial source of these compounds. To date, no method has been published that efficiently extracts and purifies capsinoids from Capsicum fruit in a rapid and simple bulk process. ARS researchers in Oxford, Mississippi, evaluated the efficacy of various organic solvents for the extraction of capsinoids from dried pepper fruit. Among the organic solvents evaluated, pentane appeared to provide a good combination of both recovery and purity. A subsequent liquid/liquid extraction step, utilizing pentane and acetonitrile, resulted in 26.3 % (wt/wt) capsiate and 19.4 % (wt/wt) dihydrocapsiate for a combined capsinoids yield of 45.7 % (wt/wt). This method and the associated patent provide a process to be utilized in producing ample quantities of capsiate for a growing market.

2. Antidiabetic activity of basil (Ocimum Species). The use of natural products as potential preventive and therapeutic interventions for diabetes has drawn worldwide attention. The Ocimum genus, collectively called basil, has been used historically as a medicinal herb for a myriad of ailments and diseases including diabetes. As part of an ongoing research project (partially funded by NIFA) to identify bioactive compound(s) associated with the anti-diabetic activity of basil, and to develop a sustainable organic production system for potential commercial cultivation, an in-depth chemical and biological study was performed by ARS researchers in Oxford, Mississippi, on sixteen Ocimum accessions consisting of six different species cultivated by Alabama A&M University (AAMU). Fourteen compounds, including terpenoids, phenolics, and flavonoids in the crude methanol extracts of the plant samples were quantitatively determined. The enzymatic inhibition activity study revealed that one accession, O.gratissimum from Zambia, strongly inhibited a-glucosidase. Moreover, two bioactive compounds, rosmarinic acid and caffeic acid that may be best associated with anti-diabetic properties of O.gratissimum species were identified. To the best of our knowledge, this is the first report of the anti-diabetic activity of these two compounds in basil species. The reported analytical and biological methods could be employed to evaluate the chemical composition and agronomic performance of Ocimum. Overall, this project provided scientific information necessary for the development of sustainable organic production systems, which enable commercial cultivation of Ocimum varieties with known bioactivity. With the right startup support, it is possible for farmers to produce a high-value crop for the national and world marketplace. A manuscript pertaining to this work has been accepted in the Journal of Food Science and Technology.

Review Publications
Zheljazkov, V.D., Cantrell, C.L., Jeliazkova, E.L., Astatkie, T., Schlegel, V. 2022. Sagebrush species of the U.S. Bighorn Mountains: essential oil content, composition, and bioactivity. Plants. https://doi.org/10.3390/plants11091228.
Akins, N.S., Mishra, N., Harris, H.M., Dudhipala, N., Kim, S., Keasling, A.W., Zjawiony, J.K., Ashpole, N.M., Le, H.V. 2022. 6,5-fused ring, C2-salvinorin ester, dual kappa and mu opioid receptor agonists as analgesics devoid of anxiogenic effects.. ChemMedChem. https://doi.org/10.1002/cmdc.202100684.
Avula, B., Bae, J., Chittiboyinaa, A.A., Wang, Y., Wang, M., Srivedavyasasri, R., Ali, Z., Li, J., Wu, C., Khan, I.A. 2021. Comparative analysis of five salvia species using LC-DAD-QToF. Journal of Pharmaceutical and Biomedical Analysis. https://doi.org/10.1016/j.jpba.2021.114520.
Maciel, G., Aparecida Lopes, A., Cantrell, C.L., De Castro Franca, S., Waleria Bertoni, B., Verginia Lourenco, M. 2021. Jasmonates promote enhanced production of bioactive caffeoylquinic acid derivative in Eclipta prostrata (L.) L. hairy roots. Plant Cell Tissue and Organ Culture. https://doi.org/10.1007/s11240-021-02201-4.
Zheljazkov, V.D., Semerdjieva, I.B., Stevens, J.F., Wu, W., Cantrell, C.L., Yankova-Tsvetkova, E., Koleva-Valkova, L.H., Stoyanova, A., Astatkie, T. 2021. Phytochemical investigation and reproductive capacity of the Bulgarian endemic plant species Marrubium friwaldskyanum Boiss. (Lamiaceae). Journal of Agricultural and Food Chemistry. https://doi.org/10.3390/plants11010114.
Cantrell, C.L., Jarret, R.L. 2022. Bulk process for enrichment of capsinoids from capsicum fruit. Processes. 10(2):305. https://doi.org/10.3390/pr10020305.
Wang, M., Cantrell, C.L., Matthews, S.T., Paudel, P., Lee, J., Mentreddy, S.R. 2022. Agronomy, chemical analysis, and antidiabetic activity of basil (Ocimum species). Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acsfoodscitech.2c00100?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as.
Zheljazkov, V.D., Semerdjieva, I.B., Cantrell, C.L., Astatkie, T., Acimovic, M. 2022. Phytochemical variability of essential oils of two Balkan endemic species Satureja pilosa Velen. and S. kitaibelii Wierzb. ex Heuff. (Lamiaceae). Molecules. https://doi.org/10.3390/molecules27103153.
Avula, B., Bae, J., Chittiboyina, A.G., Wang, Y., Wang, M., Zhao, J., Zulfiqar, A., Li, J., Wu, C., Khan, I.A. 2022. Chemometric analysis and chemical characterization for the botanical identification of Glycyrrhiza species (G. glabra, G. uralensis, G. inflata, G. echinata and G. lepidota) using liquid chromatography-quadrupole time of flig. Journal of Agricultural and Food Chemistry. https://doi.org/10.1016/j.jfca.2022.104679.
Shankara, V., Wang, M., Ajjarapua, S., Kolimi, P., Avula, B., Murthy, R., Khan, I., Naraisimha, M. 2021. Analysis of docosanol using GC/MS: Method development, validation, and application to ex vivo human skin permeation studies. Journal of Pharmaceutical and Biomedical Analysis. https://doi.org/10.1016/j.jpha.2021.08.004.
Zhao, J., Wang, M., Saroja, S.G., Khan, I.A. 2021. NMR technique and methodology in botanical health product analysis and quality control. Journal of Pharmaceutical and Biomedical Analysis. https://doi.org/10.1016/j.jpba.2021.114376.
Ameen, G., Solanki, S., Sager-Bittara, L., Richards, J., Tamang, P., Friesen, T.L., Brueggeman, R.S. 2021. Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability. PLoS Genetics. 17(12). Article e1009473. https://doi.org/10.1371/journal.pgen.1009473.
Kumairhamy, M., Ilias, M., Siddharth, T.K., Shabana, K. 2021. Schottiin, a new prenylated isoflavones from Psorothamnus schottii and antibacterial synergism studies between methicillin and fremontone against methicillin-resistant Staphylococcus aureus ATCC 1708. Natural Product Research. https://doi.org/10.1080/14786419.2021.1937157.
Sparks, T.C., Duke, S.O. 2021. Structure simplification of natural products as a lead generation approach in crop protection compound discovery. Journal of Agricultural and Food Chemistry. 69(30):8324-8346. https://doi.org/10.1021/acs.jafc.1c02616.
Sarfare, S., Khan, S.I., Zulfiqar, F., Radhakrishnan, S., Ali, Z., Khan, I.A. 2022. Undescribed C-Glycosylflavones from corn silk and potential anti-inflammatory activity evaluation of isolates. Planta Medica. https://doi.org/10.1055/a-1728-1347.
Avula, B., Katragunta, K., Wang, Y., Upton, R., Khan, I.A. 2022. Analysis of Crocetins and Saffanal variations in Saffron(Crocus sativus) stigma samples and dietary supplements using HPLC-UHPLC-PDA-MS: Chemical profiling and chemometric analysis using LC-QToF. Food Analytical Methods. https://doi.org/10.1007/s12161-022-02268-5.
Avula, B., Katragunta, K., Wang, Y., Ali, Z., Khan, I.A. 2022. Simultaneous determination and characterization of flavonoids, sesquiterpene lactone, and other phenolics from centaurea benedicta and dietary dupplements using UHPLC-PDA-MS and LC-DAD-QToF. Journal of Pharmaceutical and Biomedical Analysis. https://doi.org/10.1016/j.jpba.2022.114806.
Avula, B., Katragunta, K., Wang, Y., Ali, Z., Srivedavyasasri, R., Gafner, S., Slimestad, R., Khan, I.A. 2022. Chemical profiling and UHPLC-QToF analysis for the simultaneous determination of anthocyanins and flavonoids in Sambucus berries and authentication and detection of adulteration in elderberry dietary supplements using UHPLC-PDA-MS. Journal of Pharmaceutical and Biomedical Analysis. https://doi.org/10.1016/j.jfca.2022.104584.
Balkisu, A., Fadime, A., Zulfiqar, F., Zhao, J., Khan, I. 2022. A novel cyclohexane carboxylic acid derivative from Black Turtle Bean (Phaseolus vulgaris L.). Records of Natural Products. http://doi.org/10.25135/rnp.325.2202.2361.
Da Cruz-Silva, C.T., Cantrell, C.L., Nobrega, L.P., Ali, A., Duke, S. 2021. Bioassay-guided isolation of phytotoxins from three salvia species. Allelopathy Journal. 54(1):13-24. https://doi.org/10.26651/allelo.j/2021-54-1-1344.
Min, L., Wang, H., Bajsa Hirschel, J.N., Yu, C., Wang, B., Yao, M., Han, L., Cantrell, C.L., Duke, S.O., Sun, N., Liu, X. 2022. Novel dioxolane ring compounds for the management of phytopathogen diseases as ergosterol biosynthesis inhibitors: synthesis, biological activities and molecular docking. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.2c00541.
Cárdenas, D.M., Bajsa Hirschel, J.N., Cantrell, C.L., Rial, C., Varela, R.M., Molinillo, J.G., Macías, F.A. 2022. Evaluation of the phytotoxic and antifungal activity of C17-sesquiterpenoids as potential biopesticides. Pest Management Science. https://doi.org/10.1002/ps.7042.
Huijuan, Q., Lin, K., Li, X., Ou, H., Tan, Y., Wang, M., Wei, N. 2021. Chemical constituents and anti-gastric ulcer activity of essential oils of Alpinia officinarum (Zingiberaceae), Cyperus rotundus (Cyperaceae), and their herbal pair. Chemistry and Biodiversity. https://doi.org/10.1002/cbdv.202100214.
Kamau, R.W., Midiwo, J.O., Mgani, Q.A., Masila, V.M., Kumarihamy, M., Wang, M., Zhao, J., Muhamma, I. 2022. A new gnidiflavanone-flavonol dimer and other constituents from Gnidia apiculata. Natural Product Research. https://doi.org/10.1080/14786419.2022.2062349.
Chae, H., Dale, O., Maqbool, M., Zhao, J., Avula, B., Khan, I.A., Khan, S.I. 2022. A multitarget approach to evaluate the efficacy of Aquilaria sinensis flower extract against metabolic syndrome. Molecules. https://doi.org/10.3390/molecules27030629.
Lee, J., Wang, M., Jianping, Z., Avula, B., Chittiboyinaa, A.G., Khan, I.A. 2022. Chemical authentication and speciation of Salvia botanicals: an investigation utilizing GC/Q-ToF and chemometrics. Foods. https://doi.org/10.3390/foods11142132.
Ravu, R., Jacob, M.R., Khan, S.I., Wang, M., Cao, L., Agarwal, A.K., Clark, A.M., Li, X. 2021. Synthesis and antifungal activity evaluation of phloeodictine analogues. Journal of Natural Products. 84,2129-2137. https://doi.org/10.1021/acs.jnatprod.1c00116.