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

Location: Natural Products Utilization Research

2021 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 first year of research for this new 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 3 researcher project hired two scientists, a plant pathologist and an additional research chemist, during this year. 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. Progress Report will be divided into sections based on in-house and Non Assistant Cooperator Agreement (NACA). As part of the antifungal discovery subobjective, the new plant pathologist has been developing an improved high throughput screening (HTS) bioassay. This method is very effective, time-efficient, and provides quantitative data as well, over the traditional bioautography utilized in the past. We are optimizing and developing the HTS assay protocol for the targeted fungi. In our search for antifungal compounds from natural sources, the ethanol extract of the roots of Gentiana crassicaulis showed potent in vitro antifungal activities against the fungal pathogens Crypococcus neoformans and Aspergillus fumigatus. Given the fact that our previous antifungal screening of plant extracts generated an extremely low hit rate against A. fumigatus, this extract appeared to be a good subject for follow-up bioassay-guided fractionation. In this study, the bisphosphocholine compound irlbacholine was identified as the primary constituent of G. crassicaulis responsible for the observed antifungal activity, along with three new irlbacholine analogues, gentianalines A, B and C. Identification of the bisphosphocholines, a rare class of antifungal natural products, in these medicinal plants provides scientific evidence to complement their medicinal use. Nicotinic acid (niacin or vitamin B3) is a natural product first extracted from tobacco (Nicotiana spp.) and its derivatives can be found in many plants, such as tobacco, Areca catechu, seeds of Ricinus communis, and seed coats of cereal grains. Niacin contains an important pyridine motif and derivatives of such have been used in the synthetic design of agrochemicals, with many pesticides containing this pyridine motif such as the herbicide nicosulfuron. Fifteen niacin derivatives were synthesized in the aim of uncovering a new leading compound with phytotoxic activity. Some of the compounds displayed excellent herbicidal activity against both bentgrass and duckweed. A novel furanocoumarin was isolated and identified as a phytotoxic constituent from Amyris elimifrea a plant in the Rutaceae family and determined he absolute stereochemistry of the asymmetric center by X-ray diffraction spectroscopy. We have also refined the structure of a previously published furanocumarin isolated form a Rutaceae plant. Under the Subobjective 1.3, we have investigated a plant pathogenic fungus infecting dollarweed, a weed predominantly found in golf courses. While isolating the fungus we also isolated a bacterium associated with it. These two microbes were isolated and identified by DNA analysis. The fungus was identified as a Diaporthe species and cultured in potato-dextrose broth (PDB) in the presence and absence of Dianion HP-20. We observed about threefold increase in metabolite production when using Dianion HP-20 compared to the culturing without the resin. Bioassay guided fractionation led to the isolation of two known azaphilones and a novel azaphilone with antifungal activity against Colletotrichum species. The bacterium was identified as a Pantoea species and we have demonstrated it to have plant growth promoting activity when tested in Lemna, cucumber and sorghum plants. This bacterium is being evaluated as a fertilizer substitute for organic farming at Marrone Bio Innovations (MBI) in Davis, California, and we have signed an MTA with MBI. 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. Concluding 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. Weed interference in sugarcane (Saccharum officinarum L.) can reduce crop yield by up to 97%. However, interference is not a phenomenon caused only by weeds, as the crop has the potential to limit the growth and development of weeds. Allelopathy is one of the factors involved in interference between plants. Allelopathy is the chemical inhibition of one plant by another, due to the release into the environment of phytotoxic compounds that inhibit germination and/or growth. Plants may adversely affect growth and development of each other through the production and release of compounds into the environment. Sugarcane cultivars that are currently planted are the result of genetic improvement focused on increased crop yield. However, this selection and genetic alteration reduced the competitive potential of sugarcane, as well as its allelopathic capabilities. Many members of the Poaceae family are highly allelopathic. Thus, the objective of this study was to characterize the allelopathic potential of two sugarcane cultivars (CTC 2 and IAC 91109) by bioassay-guided isolation and identification of potential allelochemicals. For both leaves and roots, alpha-linolenic and linoleic acid were found to be the most phytotoxic compounds found with this approach. NACA with University of Mississippi: The researchers continued to source plant materials for screening from our plant collections and numerous collaborators. Eight hundred eight plant samples were added to our inventory this year and over 2,500 natural product crude extracts, semi-purified fractions, and purified compounds were screened for biological activities against specific molecular targets and whole-cell systems. As part of our continuing effort in the search for anti-infective, cancer chemopreventive, and immunomodulatory/anti-inflammatory leads from natural sources, more than 40 compounds were isolated from 11 extracts, with three showing promising activity against drug-resistant Enterococci, Streptococci, and Staphylococci strains and two exhibiting activity against Plasmodium falciparum, sans cytotoxicity to mammalian cells. Also, 16 compounds were tested against a panel of transcription factors involved in cancer signaling pathways, of which 4 had potent anticancer activity. New collaborations with the countries of Kazakhstan, Egypt, Nigeria, and Kenya were implemented for the acquisition of several plant sources. Among these extracts, 15 purified phytochemicals were tested for antimicrobial and antimalarial activity, with two exhibiting good antifungal activity as well as efficacy towards methicillin-resistant Staphylococcus aureus (MRSA). A new project has been initiated to identify pure compounds and extracts that could inhibit the binding of the spike protein of SARS-CoV-2 and ACE2, the host receptor protein. The binding of spike protein and ACE2 receptor has been reported as the pathogenic mechanism for virus entry into the cell. More than 60 pure compounds and 15 extracts were screened at various concentrations through this assay. This project aims to identify the mechanism of action of these compounds/extracts for the inhibition of COVID-19 infection, and the confirmed hits will be subjected to various mechanistic testing. From the 100 plant extracts screened for their antidiabetic and cardiovascular pharmacological activity, during the previous funding cycle, 13 were studied further to determine their adipogenic, antiadipogenic, and glucose uptake effects. Their effect on insulin sensitivity and glucose transport as well as lipid metabolism was determined through Liver X Receptor (LXR) agonism assays. Ten medicinal plants were screened for anti-inflammatory activity mediated through modulation of two pathways, namely inducible nitric oxide synthase activity, which leads to excessive generation of intracellular nitrates, and activation of the NRF2-ARE signaling pathway, which regulates the cellular antioxidant response. High throughput cell-based assays utilized for this purpose employed mouse macrophage (RAW 264.7) and human hepatic (HepG2) cell lines. In addition to the above scientific activities, the Medicinal Plant Garden at the University of Mississippi continues to expand its renowned collection of living medicinal plants. New demonstration beds and field plots were developed. Here NCNPR cultivates and processes medicinal plants to be used in the discovery program, emphasizing new acquisitions from other countries. The new research wing will expand and enhance the research capabilities of NCNPR with a second plant specimen repository, herbarium, and laboratories for plant tissue cultures, cellular cultures, scale-up isolation, and synthetic chemistry.

Accomplishments
1. A novel approach for lavender essential oil authentication and quality assessment. Lavandula angustifolia Mill., commonly known as English lavender or true lavender, is an important agricultural plant which has been used to produce lavender essential oil (LEO). The oil is one of the most frequently adulterated essential oils, primarily due to the plants low oil production and high commercial value. Currently, the quality of LEO is defined and regulated based on the standards and methods established by various authoritative organizations. Due to the complexity of LEO, these existing standards and methods are not sufficient for quality control and to protect LEO from adulteration. The goal for ARS researchers in Oxford, Mississippi, was to develop an efficient and reliable method for LEO quality assessment and adulteration detection. After a comprehensive investigation, involving a large set of LEO samples analyzed by multiple techniques (GC/MS, GC/Q-ToF, NMR, and chemometric analysis), a new approach named Q-Index was proposed. Q-Index provided superior results compared to the ISO method, and successfully detected poor- quality LEO and LEO adulterated by addition of lavandin oil, ho leaf oil, cooking oil, and synthetic compounds. The results from this study demonstrated the feasibility and simplicity of the proposed Q-Index method as an efficient and reliable tool for routine analysis and quality control of LEOs, the international herbal products, cosmetic, and essential oil industries, and the extended natural products community in general.

2. Natural products as antimicrobial agents with a novel mechanism of actions. In an ongoing effort to develop antimicrobial agents from natural products, ARS researchers in Oxford, Mississippi, performed studies in collaboration with Kings College London, United Kingdom, on the inhibitory activities and synergism studies of machaeriols and machaeridiols against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium. In addition, they were evaluated against permeabilized Gram-negative pathogens, which showed promising leads. A grant pre-proposal, followed by a full proposal, was submitted to the Wellcome Trust, United Kingdom.

3. Identify dietary or botanical products with cancer chemopreventive potential. This program identifies the most promising lead natural product extracts/pure compounds, using our battery of 15 cancer signaling pathway luciferase assays, to determine their cytotoxic potential against various cells in vitro and characterizes their mechanism(s) of action. A potent formulation with a unique mechanism of action containing a mixture of pure compounds and extracts has been identified through this extensive screening process. The PCT application that was filed last year on this formulation entered into a national phase, and a patent application has been filed. Preclinical studies are underway. Molecular assays have been carried out by ARS researchers in Oxford, Mississippi, to identify the enzyme(s) responsible for their mechanism of action. These enzymes could be possible molecular targets and could also serve as biomarkers when advancing to translational studies. Various combinations of this formulation have been tested in murine colon cancer models to establish its in vivo potential. Both biological and analytical methods have been developed to evaluate the pharmacokinetic properties of this formulation within mice, and target organs have been identified from bio-distribution patterns. To maximize the solubility and to increase bioavailability, various modifications of pure compounds were tested for intravenous administration. Optimal efficacy in target organs and maximum tolerated dosage studies are ongoing. This formulation might serve as a candidate for translational research and product development. To overcome the supply issue of the test article, an efficient synthetic strategy has been proposed and is being evaluated for feasibility. Thirty-five herbal extracts/pure compounds were also evaluated in various cancer cell lines (glioblastoma, colon cancer, and leukemia) for their synergistic interactions.

Review Publications
Perera, W.H., Meepagala, K.M., Duke, S.O. 2020. Sesquiterpene-a-amino acid quaternary ammonium hybrids from Stereum complicatum (Steraceae). Biochemical Systematics and Ecology. 93,104176. https://doi.org/10.1016/j.bse.2020.104176.
Eliwa, D., Albadry, M.A., Ibrahim, A.S., Kabbash, A., Meepagala, K.M., Khan, I.A., El-Aasr, M., Ross, S.A. 2020. Biotransformation of papaverine and in silico docking studies of the metabolites on human phosphodiesterase 10a. Phytochemistry. https://doi.org/10.1016/j.phytochem.2020.112598.
Zhai, C., Zhao, J., Chittiboyina, A.G., Meng, Y., Wang, M., Khan, S.I., Khan, I.A. 2020. Newly generated Atractylon derivatives in the processed rhizomes of Atractylodes macrocephala Koidz. Molecules. https://doi.org/10.3390/molecules25245904.
Wei, N., Wang, M., Adams, S.J., Yu, P., Avula, B., Wang, Y., Pan, K., Wang, Y., Khan, I.A. 2020. Comparative study and quality evaluation regarding morphology characters, volatile constituents and triglycerides in seeds of five species used in traditional Chinese medicine. Journal of Pharmaceutical and Biomedical Analysis. 194. Article e113801. https://doi.org/10.1016/j.jpba.2020.113801.
Wang, M., Zhao, J., Ali, Z., Avonto, C., Khan, I. 2021. A novel approach for lavender essential oil authentication and quality assessment. Journal of Pharmaceutical and Biomedical Analysis. 199. Article e114050. https://doi.org/10.1016/j.jpba.2021.114050.
Muhammad, I., Jacob, M.R., Ibrahim, M.A., Raman, V., Kumarihamy, M., Wang, M., Al-Adhami, T., Hind, C., Clifford, M., Martin, B., Sutton, M., Rahman, M., Zhao, J. 2020. Antimicrobial constituents from Machaerium Pers.: Inhibitory activities and synergism of machaeriols and machaeridiols against methicillin-resistant Staphylococcus aureus and Vancomycin-resistant Enterococcus faecium and perm. Molecules. 25/6000. https://doi.org/10.3390/molecules25246000.
Balachandran, P., Ibrahim, M.A., Zhang, J., Wang, M., Pasco, D.S., Muhammad, I. 2021. Cross talk of cancer signaling pathways by cyclic hexapeptides and anthraquinones from Rubia cordifolia. Molecules. 26/735. https://doi.org/10.3390/molecules26030735.
Avula, B., Bae, J., Zhao, J., Wang, Y., Wang, M., Zhang, Z., Ali, Z., Khan, I. 2021. Quantitative determination and characterization of polyphenols from Cissus quadrangularis L. and dietary supplements using UHPLC-PDA-MS, LC-QToF and HPTLC. Journal of Pharmaceutical and Biomedical Analysis. https://doi.org/10.1016/j.jpba.2021.114036.
Abdelmalek, E.M., Zulfiqar, F., Albadry, M.A., Khan, S.I., Meepagala, K.M., Ramadan, M.A., Darwish, F.M., Assaf, M.H., Ross, S.A. 2021. In silico and in vitro studies of isolated constituents from Callistemon citrinus leaves: Anti-microbial potential and inhibition of iNOS activity. Phytochemistry. 185. https://doi.org/10.1016/j.phytochem.2021.112745.
Barreto, D.L., Nogueira De Azevedo, R., Carvalho, C., Ferreira, M.C., Cantrell, C.L., Duke, S.O., Rosa, L.H. 2021. Bioactive compounds produced by Neotropical endophytic fungi applied to agriculture. In: Rosa L.H. editors. Neotropical Endophytic Fungi. Springer, Cham: Springer International Publishing. p. 257-295.
Meepagala, K.M., Bracken, A.K., Fronczek, F.R., Johnson, R.D., Wedge, D.E., Duke, S.O. 2020. A novel furanocoumarin with phytotoxic activity from the leaves of Amyris elemifera (Rutaceae). Journal of Agricultural and Food Chemistry. https://www.doi.org/10.1021/acsomega.0c04778.
Ren, S., Deng, K., Qiu, S., Wang, M., Avula, B., Tripathi, S.K., Jacob, M.R., Gong, L., Wang, W., Khan, I.A., Li, X. 2020. Identification of antifungal Bisphosphocholines from medicinal Gentiana species. Journal of Natural Products. 2020, 83, 10, 3207–3211. https://doi.org/10.1021/acs.jnatprod.0c00584.
De Oliveira, T.L., Fontana, P.D., Bavia, L., Cruz, L., Crisma, A.R., Sassaki, G.L., Menezes, L.R., Wang, M., Beltrame, F., Messias-Reason, I.J. 2020. Effects of Euphorbia umbellata extracts on complement activation and chemotaxis of neutrophils. Journal of Ethnopharmacology. 265/113348. https://doi.org/10.1016/j.jep.2020.113348.
Avula, B., Parveen, I., Zhao, J., Wang, M., Techen, N., Wang, Y., Riaz, M., Bae, J., Shami, A.A., Chittiboyina, A.G., Khan, I.A., Sharp, J.S. 2021. A comprehensive workflow for the analysis of Bio-Macromolecular supplements: Case Study of 20 Whey protein products. Journal of Dietary Supplement. https://doi.org/10.1080/19390211.2021.1897724.
Avula, B., Bae, J., Wang, Y., Wang, M., Ali, Z., Khan, I.A. 2021. Chemical profiling and characterization of anthraquinones from two Bulbine species and dietary supplements using liquid chromatography-high resolution mass spectrometry. Nature Product Communications. https://doi.org/10.1093/jaoacint/qsab075.
Stefenoni, H., Räisänen, S., Cueva Welchez, S., Wasson, D., Lage, C., Melgar, A., Fetter, M., Smith, P., Hennessy, M., Vecchiarelli, B., Bender, J., Pitta, D., Cantrell, C.L., Yarish, C., Hristov, A. 2021. Effects of the macroalga Asparagopsis taxiformis and oregano leaves on methane emission, rumen fermentation, and lactational performance of dairy cows. Journal of Dairy Science. 104:4157-4173. https://doi.org/10.3168/jds.2020-19686.
Yu, C., Wang, Q., Min, L., Bajsa Hirschel, J.N., Hua, X., Cantrell, C.L., Duke, S., Liu, X. 2021. Synthesis, crystal structure, herbicidal activity, and SAR study of Novel N-(Arylmethoxy)-2-chloronicotinamides derived from nicotinic acid. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.0c07538.
Zheljazkov, V.D., Cantrell, C.L., Semerdjieva, I., Radoukova, T., Stoyanova, A., Maneva, V., Kacaniova, M., Astatkie, T., Borisova, D., Dincheva, I., Salamon, I. 2021. Essential oil and bioactivity of two juniper species from Bulgaria and Slovakia. Molecules. 2021, 26, 3659.
Hijano, N., Nepomuceno, M.P., Cantrell, C.L., Duke, S.O., Alves, P.L. 2021. Characterization of the allelopathic potential of sugarcane leaves and roots. Journal of Agricultural Chemistry and Environment. https://doi.org/10.4236/jacen.2021.103016.
Hawwal, M.F., Ali, Z., Wang, M., Zhao, J., Lee, J., Fantoukh, O.I., Khan, I.A. 2021. (E)-2,6,10-Trimethyldodec-8-en-2-ol: An undescribed sesquiterpenoid from copaiba oil. Molecules. 2021, 26, 4456. https://doi.org/10.3390/molecules26154456.
Avonto, C., Chittiboyina, A.G., Khan, S.I., Dale, O.R., Parcher, J.F., Wang, M., Khan, I.A. 2020. Are atranols the only skin sensitizers in oakmoss, a systematic investigation using non-animal methods. Toxicology In Vitro. 70. Article e105053. https://doi.org/10.1016/j.tiv.2020.105053.
Shaukat, U., Ahemad, S., Wang, M., Khan, S.I., Ali, Z., Muhammad I, T.I., Abdallah, H.H., Khan, I.A., Saleem, M., Mahomoodally, M.F. 2021. Phenolic contents, chemical profiling, in silico and in vitro antiinflammatory and anticancer properties of Alnus nitida (Spach) Endl. South African Journal of Botany. 138:148-155. https://doi.org/10.1016/j.sajb.2020.12.010.