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ARS Home » Southeast Area » Oxford, Mississippi » Natural Products Utilization Research » Research » Research Project #428996

Research Project: Discovery and Development of Natural Products for Pharmaceutical and Agrochemical Applications II

Location: Natural Products Utilization Research

2020 Annual Report

Objective 1: Discover new leads from plant-based natural resources with anti-infective and anti-cancer, immunomodulator and anti-inflammatory activities using cell-based screens and mechanistic assays using molecular target-based approaches. Sub-objective 1A: Source novel natural resources from terrestrial plants, marine organisms and microbes from around the world for biological testing. Sub-objective 1B: Prepare, maintain and manage Natural Products Repository and Laboratory Information Management System (LIMS). Sub-objective 1C: Evaluate natural product extracts and natural product derived pure compounds for potential anticancer, anti-infective, anti-inflammatory, neuroprotective and anti-diabetic properties, and for utility in metabolic and immune disorders. Sub-objective 1D: Isolation and structural elucidation of lead compounds. Objective 2: For the best candidates, characterize mechanisms of action, selectivity, toxicity, functional activity in secondary assays and in animal models of plant-based anti-infective and anti-cancer, immunomodulator and anti-inflammatory compounds. Sub-objective 2A: Characterization of mechanisms of action and functional activity of leads. Sub-objective 2B: Characterization of selectivity and toxicity of lead compounds. Objective 3: Develop methods for analysis of bioactive or medicinally important plants and quality control of their derived products. Objective 4: Assess selected medicinal or aromatic plants for cultivation, harvest and processing to optimize yields of biomass and active principles.

The approach includes a program of: (1) Using cell-based screening and mechanistic assays to discover new pharmaceutical and agrochemical leads from natural sources; (2) Using secondary assays and animal models to characterize mechanisms of action, selectivity, toxicity and functional activity of the best candidate compounds having anti-infective, anti-cancer, immunomodulatory, or anti-inflammatory properties; (3) Selection, agronomics and analysis of bioactive or medicinally important plants and their derived products.

Progress Report
This is the final report for this project, replaced with bridging project #6060-41000-015-00D pending completion of National Program 306 research review. The overall outcome and summary of extensive research efforts were undertaken within the last 5 years (reporting cycle) project at the National Center for Natural Products Research (NCNPR) at the University of Mississippi, Oxford, Mississippi, is delineated below. The team at the NCNPR maintained basic discovery operations, with emphasis on the discovery of anti-fungal, anti-cancer, anti-inflammatory agents, and immunomodulating agents. The researchers continued to source plant materials for screening from our plant collections and numerous collaborators. Including this year’s 955, a total of > 4,400 plant samples were added to our inventory for this reporting cycle. In addition to the collection of a wide variety of plants, isolates originated from other sources such as yeast and bacterial were also collected and allowed as prepare > 3,000 extracts and over 35,000 semi-purified fractions, and purified compounds were screened for biological activities against specific molecular targets and whole-cell systems. Based on preliminary screening data, close to 75 isolation projects were undertaken to prepare more than 800 purified natural products and their synthetic analogs (250). Specifically, extracts, enriched fractions of biologically active compounds further screened for promising anti-infective, cancer chemopreventive, and immunomodulatory/anti-inflammatory properties. Many of the fully characterized pure components and their analogs were evaluated mechanistically as standalone or in combination with known antifungal drugs for the possible potentiation effects in animal models. Genomic and biochemical approaches allowed us to identify new biological pathways associated with natural products as antifungal agents. Several natural products were identified to unmask ß-glucan from fungal cells and could serve as antifungal agents with a novel mechanism of action. To explore the antidiabetic and cardiovascular potential of medicinal plants, for this reporting period, about 380 plant extracts and 315 pure compounds were screened for peroxisome proliferator-activated receptor (PPAR)-alpha and gamma agonistic activity. Many active extracts were selected and were followed further in secondary assays such as adipogenic effect, antiadipogenic effect, and the agonistic effect towards liver X receptor (LXR) (which regulates lipid metabolism). The immunomodulatory effects of terrestrial plants and microbes were also investigated under this cooperative agreement as an innovative potential of natural products. In addition to these basic operations, we have selected a number of these compounds for more advanced study, whether for characterizing mechanisms of action, determining suitability for further pharmaceutical development, evaluation in disease models in preclinical studies, or field applications. Close to 575 botanical extracts and 390 pure compounds were screened for anti-inflammatory activities mediated via nitric oxide synthase and NRF2-ARE signaling pathways. At the same time, many of these extracts and pure compounds were screened for anti-proliferative effects in various human cancer cell lines. A non-cancer kidney cell line was also included to compare the efficacy and to determine the selectivity. Another safety aspect we have undertaken for this activity was to gauge the potential herb-drug interactions of medicinal plants, a collection of the plant extracts was also screened for their potential for causing drug interaction by affecting the pharmacokinetics of concurrently consumed drugs due to changes in the drug-metabolizing enzymes. The screening was performed through high throughput assays for the activation of Pregnane X Receptor (PXR) and inhibition of Cytochrome P450 (CYP) 3A4 isoform. As a result, more than 200 plant extracts were tested in a cell-based assay to gauge both activation and inhibitory effects on cytochrome P 450s and confirmatory experiments with enzymatic activity and gene expression. 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. 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.

1. Agents for the prevention/treatment of poison ivy dermatitis. University researchers along with ARS researchers in Oxford, Mississippi, in collaboration with ElSohly Laboratories, Inc. are developing preventive treatments for poison ivy dermatitis. Development continued on the compound shown to be effective in animal models for desensitization to poison ivy dermatitis and shown to have desirable bioavailability and toxicological properties. This product has been given the investigational new drug (IND) status by the U.S. Food and Drug Administration.

2. Natural antimicrobial agents with a novel mechanism of action. Under an ongoing research collaboration with King’s College London, United Kingdom and Public Health England, an advanced in vitro, ex-vivo, mechanistic, and resistance reversal studies against a panel of microbes resistant to antibiotics were carried out on selected lead machaeriols and their analogs. These compounds were previously isolated and analogs prepared by university researchers in collaboration with ARS researchers in Oxford, Mississippi.

3. Natural immunomodulators to suppress the pathogenic effects of influenza virus infections. A new animal model has been established to study the effect of the plant-based immunomodulatory product(s) for effectiveness on minimizing the pathogenic effects of influenza virus infection by the university researchers in collaboration with ARS researchers in Oxford, Mississippi. Pneumonic lesions in the lung after influenza virus infection in mice have been characterized. The influenza virus from the infected lung was successfully titrated and its pathogenicity in cell culture has been established.

4. Botanical products with cancer chemopreventive potential. The university researchers in collaboration with ARS researchers in Oxford, Mississippi, identified several promising lead natural product extracts/pure compounds as a chemopreventive agents using battery of 15 cancer signaling pathway luciferase assays, and determines their cytotoxic potential against both bulk and tumor stem-like cells isolated from various types of patient-derived tumor biopsies in vitro and characterizes their mechanism of action. As a result, a PCT application has been filed on the most promising formulation, identified a possible molecular target, and studied with mouse melanoma models to establish the in vivo efficacy. Both biological and analytical methods have been developed to evaluate the pharmacokinetic properties of this formulation with mouse models and target organs have been determined from their bio-distribution pattern. 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 being evaluated for feasibility. Several herbal extracts/pure compounds synergistic interactions (about 20 combinations) were also evaluated in various cancer cell lines (glioblastoma, triple-negative breast cancer, and melanoma). Effective combinations were screened further in bulk and tumor stem-like cells (isolated from various types of patient-derived tumor biopsies) in vitro.

5. Characterization and development of immunomodulatory natural products. For over 20 years, a major focus of university researchers in collaboration with ARS researchers in Oxford, Mississippi, has been the identification of components responsible for the immune-enhancing and immune-inhibitory properties of botanicals and plants. This research has resulted in the development of a novel theory that the naturally occurring bacterial communities within plants produce potent activators of pathogen recognition receptors that are principal contributors in the activation of innate immune cells. Building on this foundation, a project was completed on the characterization of the immune-enhancing properties of mushrooms. Results demonstrated that the macrophage stimulatory activity of edible mushrooms is due to the collaborative interaction of water-soluble Toll-like receptor agonists (derived from colonizing bacteria) and water-insoluble particulate ß-glucans (derived from colonizing yeast). Continued efforts support the development of the patented extract, ImmulinaTM (commercially available as a dietary supplement), to promote resilience against and/or recovery from respiratory viral infections. This extract was discovered by the university researchers and ARS researchers at Oxford, Mississippi, and it is enriched with the active immunostimulatory compounds in Arthrospira (Spirulina) platensis, i.e., Braun-type lipoproteins (potent Toll-like receptor 2 agonists). A research service center continues to perform biological standardization of this botanical product. Screening efforts resulted in three focused projects: characterization of soluble ß-glucans (activators of dectin-1a) from plants; isolation of Toll-like receptor 4 (TLR4) activators from black beans (identified as the known compound, phytohemagglutinin); and identification of cyanobacteria that contains a compound exhibiting selective inhibition of the TLR4 signaling pathway.

6. Antifungal natural products for use in agriculture and medicine. This interim, research project has continued to two separate projects. The first project is focused on the identification of compounds that potentiate current antifungal drugs with the long-term goal of developing new combination therapies for fungal infections. The second project aims to identify compounds that unmask ß-glucan from fungal cells, thereby allowing their clearance due to their exposure to human immune cells. This project has applications in the treatment of fungal infections associated with metabolic diseases such as diabetes, and also in the management of irritable bowel disease which is aggravated by gut-residing fungi. In the first project, over 700 compounds were assayed for their ability to potentiate the activity of the antifungal drug caspofungin (CAS), which inhibits fungal cell wall synthesis, and over 25 candidates were identified. In the previous project period, university researchers in collaboration with ARS researchers in Oxford, Mississippi, had identified several compounds that potentiated CAS activity, and in this interim, RNA sequencing analysis on three compounds revealed that they induced the cell wall repair pathway in fungal cells. This study indicates that when CAS is combined with a compound that induces cell wall repair pathways, fungal growth is severely inhibited because the cells are unable to cope with the cell wall dysfunction exerted by the two agents. In the second project, seven compounds have been identified that have the potential to unmask ß-glucan from fungal cells. In this interim, a mechanistic assay on two of the compounds revealed that they altered the sensitivity of fungal cells to the enzyme ß-glucanase, further confirming that the two compounds play a role in unmasking ß-glucan. Follow-up mechanistic and animal studies are planned for the most promising compounds from both projects.

Review Publications
Bae, J., Avula, B., Wang, Y., Wang, M., Ali, Z., Viljeon, A.M., Khan, I.A. 2019. Development and validation of a UHPLC-PDA-MS method for the quantitative analysis of anthraquinones in Bulbine natalensis extracts and dietary supplements. Planta Medica. 86:144-150.
Yalamanchili, C., Chittiboyina, A.G., Haider, S., Vasquez, Y., Khan, S., Do Carmo, J.M., Da Silva, A.A., Pinkerton, M., Hall, J.E., Walker, L.A., Khan, I.A. 2020. In search for potential antidiabetic compounds from natural sources: docking, synthesis and biological screening of small molecules from Lycium spp. (Goji). Heliyon. 6(1):e02782.
Manda, V., Haron, M.H., Mir, T.M., Avula, B., Ashfaq, M.K., Haider, S., Chittiboyina, A.G., Khan, I.A., Khan, S.I. 2019. Pharmacokinetics and tissue distribution of Aegeline after oral administration in mice. Planta Medica. 85(06):491-495.
Ajayi, T.O., Srivedavyasasri, R., Nyong, E.E., Odeniyi, M.A., Moody, J.O., Ross, S.A. 2019. Two new phytoecdysteroids from Sphenocentrum jollyanum Pierre root. Steroids. 150:108456.
Labib, R.M., Zulfiqar, F., Ibrahim, M.A., Balachandran, P., Zhang, J., Ross, S.A. 2019. FOXO signal activating alkaloids isolated from Ochrosia elliptica leaf cultivated in Egypt. Medicinal Chemistry Research. 28:1628-1632.
Zhang, J., Tyler, H.L., Haron, M.H., Jackson, C.R., Pasco, D.S., Pugh, N.D. 2019. Macrophage activation by edible mushrooms is due to the collaborative interaction of toll-like receptor agonists and dectin-1b activating beta glucans derived from colonizing microorganisms. Food & Function. 10:8208-8217.
Mir, T., Ma, G., Ali, Z., Khan, I.A., Ashfaq, M.K. 2019. Effect of raspberry ketone on normal, obese and health-compromised obese mice: A preliminary study. Journal of Dietary Supplement.
Ibrahim, M.A., Cantrell, C.L., Jeliazkova, E.A., Astatkie, T., Zheljazkov, V.D. 2020. Utilization of nutmeg (Myristica fragrans Houtt.) seed hydrodistillation time to produce essential oil fractions with varied compositions and pharmacological effects. Molecules. 25(3):565.
Masila, V.M., Ndakala, A.J., Byamukama, R., Midiwo, J.O., Kamau, R.W., Wang, M., Kumarihamy, M., Zhao, J., Heydreich, M., Muhammad, I. 2020. Synthesis, structural assignments, and antiinfective activities of 3-0-benzyl-carvotacetone and 3-hydroxy-2-isopropyl-5-methyl-p-benzoquinone. Natural Product Research.
Parveen, A., Maqbool, M., Wang, Y., Ali, Z., Khan, I.A., Ashfaq, M. 2020. Evaluation of the hepatotoxic potential of Tinospora crispa and its isolated borapetosides B, C and F in a murine model. Planta Medica. 86(07):489-495.
Wang, M., Yu, P., Chittiboyina, A.G., Chen, D., Zhao, J., Avula, B., Wang, Y., Khan, I.A. 2020. Characterization, quantification and quality assessment of Avocado (Persea americana Mill.) oils. Molecules. 25(6):1453.
Avula, B., Bae, J., Wang, Y., Wang, M., Osman, A.G., Smith, K., Yuk, J., Ali, Z., Plumb, R., Isaac, G., Khan, I.A. 2020. Chemical profiling and characterization of phenolic acids, flavonoids, terpene glycosides from Vangueria agrestis using ultra-high-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry and metabolomics approach. Biomedical Chromatography.
Kozykeyeva, R.A., Datkhayev, U.M., Srivedavyasasri, R., Ajayi, T.O., Patsayev, A.K., Kozykeyeva, R.A., Ross, S.A. 2020. Isolation of chemical compounds and essential oil from Agrimonia asiatica Juz. and their antimicrobial and antiplasmodial activities. The Scientific World.
Mohamed, N.M., Makboul, M.A., Farag, S.F., Wang, Y., Mohamed, S.M., Ross, S.A. 2020. Chemosystematicaly valuable triterpenoid saponins from Glandularia x hybrida. Phytochemistry. 175:112367.
Zulfiqar, F., Khan, S.I., Ali, Z., Wang, Y., Ross, S.A., Vilijoen, A.M., Khan, I.A. 2020. Norlignan glucosides from Hypoxis hemerocallidea and their potential in vitro anti-inflammatory activity via inhibition of iNOS and NF-kB. Journal of Natural Products. 172:112273.
Bae, J., Ali, Z., Wang, Y., Chittiboyina, A.G., Zaki, A.A., Viljeon, A.M., Khan, I.A. 2019. Anthraquinone-based specialized metabolites from rhizomes of Bulbine natalensis. Journal of Natural Products. 82:1893-1901.
Bae, J., Avula, B., Zhao, J., Raman, V., Wang, Y., Wang, M., Zulfiqar, F., Feng, W., Park, J., Abe, N., Ali, Z., Khan, I.A. 2019. Analysis of Prenylflavonoids from aerial parts of Epimedium grandiflorum and dietary supplements using HPTLC, UHPLC-PDA and UHPLC-QToF along with chemometric tools to differentiate Epimedium species. Journal of Pharmaceutical and Biomedical Analysis. 177:112843.
Qiu, S., Khan, S.I., Wang, M., Zhao, J., Ren, S., Khan, I.A., Steffek, A., Pfund, W.P., Li, X. 2020. Chemometrics-assisted identification of anti-inflammatory compounds from the green alga Klebsormidium flaccidum var. zivo (KALGAE™). Molecules. 25(5):1048.
Tripathi, S.K., Feng, Q., Liu, L., Levin, D.E., Roy, K.K., Doerksen, R.J., Baerson, S.R., Shi, X., Pan, X., Xu, W., Li, X., Clark, A.M., Agarwal, A.K. 2020. Puupehenone, a marine sponge-derived sesquiterpene quinone, potentiates the antifungal drug Caspofungin by disrupting Hsp90 activity and the cell wall integrity pathway. mSphere. 5(1):e00818-19.
Ye, X., Wang, Y., Zhao, J., Wang, M., Avula, B., Peng, Q., Ouyang, H., Lingyun, Z., Zhang, J., Khan, I.A. 2019. Identification and charaterization of key chemical constituents in processed Gastrodia elata using UHPLC-MS/MS and chemometric methods. Journal of Analytical Methods in Chemistry.