Discovery and Development of Natural Products for Pharmaceutical and Agrochemical Applications II

Goals / Objectives
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.

Project Methods
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 07/07/15 to 07/06/20

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. 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 ÿ-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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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 ÿ-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 ÿ-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. 06 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 ÿ-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 ÿ-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 ÿ-glucanase, further confirming that the two compounds play a role in unmasking ÿ-glucan. Follow-up mechanistic and animal studies are planned for the most promising compounds from both projects.

Impacts
(N/A)

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.
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.
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.
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.

Progress 10/01/18 to 09/30/19

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. Researchers at the National Center for Natural Products Research (NCNPR) at the University of Mississippi, Oxford, Mississippi maintained basic discovery operations, with emphasis on the discovery of antifungals, anticancer, anti-inflammatory agents and immunomodulating agents. The researchers continued to source plant materials for screening from our own plant collections and from numerous collaborators. We added 955 plant samples to our inventory this year and screened over 6700 natural product crude extracts, semi-purified fractions and purified compounds 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 immunomodulator/anti-inflammatory leads from natural sources, more than 100 compounds were isolated. In addition, 20 compounds were synthesized. Of the compounds tested, 21 were found to be biologically active for potential agricultural or medical uses. Many showed potent phytotoxic, antifungal, antibacterial, or antimalarial activities. In order to explore the antidiabetic potential of medicinal plants, about 30 plant extracts were screened for peroxisome proliferator-activated receptor (PPAR) agonistic activity. About 20 pure compounds were explored for antidiabetic potential. The actives 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. 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 in field applications. About 125 plant extracts were screened for anti- inflammatory activities through target-based cellular assays including Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB), Inducible Nitric Oxide Synthase (iNOS), and oxidative stress and about 50 pure compounds were screened for anti-inflammatory activities through these targets. About 25 extracts of medicinal plants and 20 isolated pure constituents from medicinal plants were also screened for their potential of causing drug interaction in terms of pregnane X receptor (PXR) activation and Cytochrome P405s (CYPs) and permeability glycoprotein (P- gp) inhibitions. In collaboration with ElSohly Laboratories, Inc., continued development of a product shown to be effective in animal models for desensitization to poison ivy dermatitis. The lead compound in the poison ivy project is now being evaluated in stage 1 clinical trials. 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. Accomplishments 01 Researchers continued with the development of agents for the prevention/ treatment of poison ivy dermatitis through cooperative agreement with ARS scientists in Oxford, Mississippi. Scientists at the University of Mississippi, 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 attained investigational new drug (IND) status by the U.S. Food and Drug Administration. 02 Natural immunomodulators to suppress the pathogenic effects of influenza virus infections. Through cooperative agreement with ARS scientists in Oxford, Mississippi, scientists at the University of Mississippi, Oxford, Mississippi, have established a new animal model to study the effect of the plant-based immunomodulatory product(s) for effectiveness on minimizing the pathogenic effect of influenza virus infection. 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. 03 Identify dietary or botanical products with cancer chemopreventive potential. Through cooperative agreement with ARS scientists in Oxford, Mississippi, this program takes the most promising lead natural product extracts/pure compounds, identified using our 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. As a result, a provisional patent 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 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. In addition, several herbal extracts/pure compounds synergistic interactions (about 20 combinations) were 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. 04 Characterization and development of immunomodulatory natural products. Through cooperative agreement with ARS scientists in Oxford, Mississippi, for over 15 years, a major focus of the scientists in this research area at the National Center for Natural Products Research (NCNPR), University of Mississippi, has been identifying the components responsible for the immune-enhancing properties of botanicals. 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, current efforts are directed towards characterization of the immune-enhancing properties of mushrooms. Data indicate that the macrophage stimulatory activity of edible mushrooms is due to the collaborative interaction of water-soluble Toll-like receptor agonists (derived from microbiome bacteria) and water-insoluble particulate beta glucans (derived from microbiome yeast). Continued efforts support the development of the patented extract, ImmulinaTM, that is commercially available as a dietary supplement for modulating innate immune function. This extract was discovered at the NCNPR 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 has been established for the biological standardization of this botanical product. Screening efforts identified a bacterium exhibiting selective inhibition of the Toll-like receptor 4 (TLR4) signaling pathway. The compound responsible was isolated using bio-assay-guided fractionation and structural characterization is currently in progress. Inflammatory conditions and disease are potential therapeutic targets for TLR4 inhibitors. 05 New antifungal natural products for use in agriculture and medicine. Through cooperative agreement with ARS scientists in Oxford, Mississippi, scientists at the National Center for Natural Products Research (NCNPR), University of Mississippi, have continued on this research effort of the identification of compounds that can synergize with current antifungal drugs with the long-term goal of developing new combination therapies to treat fungal infections. First, we have evaluated ~2500 compounds for their ability to induce the fungal cell wall damage response pathway. Compounds identified will be expected to synergize with the cell wall-targeting antifungal drug caspofungin (CAS) . Over 20 hits were identified and several were confirmed to potentiate CAS activity. Second, we have conducted dose-matrix assays to directly evaluate compounds for CAS-synergizing activity in a CAS-resistant pathogen. Over 300 compounds were evaluated, and 10 candidates showed synergistic activity. Follow-up mechanistic and animal studies are planned for the most promising compounds from both projects. We have also evaluated 14 compounds and 21 extracts for their ability to unmask beta-glucan from the cell walls of the fungal pathogen, Candida albicans. Samples identified will be expected to expose fungal cells to host immune cells, thereby allowing fungal clearance from the infection site. Given that Candida species inherently exist in the human gut, this strategy will also be useful in preventing fungal infections, most of which originate from the gut population. In addition, given the role gut-residing fungi play in irritable bowel disease (IBD), this strategy will be useful in the management of IBD. In our assay, 15 samples exhibited beta-glucan unmasking activity, and follow-up studies on them are ongoing. The most promising ones will be evaluated mechanistically, and in studies with animal models of fungal infections and IBD.

Impacts
(N/A)

Publications

Avula, B., Bae, J., Raman, V., Wang, Y., Fantoukh, O., Osman, A., Wang, M., Ali, Z., Khan, I.A. 2018. Quantification of phenolic compounds from Fadogia agrestis and dietary supplements using UHPLC-PDA-MS. Analytical and Bioanalytical Chemistry. 85(2):145-154.
Avula, B., Bae, J., Wu, T., Wang, Y., Wang, M., Majrashi, T., Ali, Z., Wu, Y., Khan, I.A. 2018. Targeted and non-targeted analysis of annonaceous alkaloids and acetogenins from Asimina and Annona species using UHPLC- QTofF-MS. Journal of Pharmaceutical and Biomedical Analysis. 159:548-566.
Haron, M.H., Dale, O.R., Zulfiqar, F., Wang, Y., Chittiboyina, A.G., Khan, I.A., Khan, S.I. 2019. Effect of African potato (Hypoxis hemerocallidea) extracts and its constituents on PXR and CYP450 Enzymes. Applied In Vitro Toxicology. 5(1):26-33.
Haron, M.H., Tyler, H.L., Chandra, S., Moraes, R.M., Jackson, C.R., Pugh, N.D., Pasco, D.S. 2019. Plant microbiome-dependent immune enhancing action of Echinacea purpurea is enhanced by soil organic matter content. Scientific Reports. 9(136):1-11.
Haron, M.H., Avula, B., Qui, S., Li, X., Ashfaq, M.K., Bae, J., Guan, S., Hinchee, M., Khan, I.A., Khan, S.I. 2019. Quantitative determination and pharmacokinetic study of fusaricidin A in mice plasma and tissues using ultra-high performance liquid chromatography-tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 170:187-192.
Li, N., Khan, S.I., Qui, S., Li, X. 2018. Synthesis and anti-inflammatory activities of Phloroglucinol-based derivatives. Molecules. 23(12):3232- 3240.
Qiu, S., Avula, B., Guan, S., Ravu, R., Wang, M., Zhao, J., Khan, I.A., Hinchee, M., Li, X. 2018. Identification of fusaricidins from the antifungal microbial strain Paenibacillus sp. MS2379 using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Journal of Chromatography A. 1586:91-100.
Rehman, J.U., Wang, M., Yang, Y., Liu, Y., Li, B., Qin, Y., Wang, W., Chittiboyina, A.G., Khan, I.A. 2019. Toxicity of Kadsura coccinea (Lem.) A. C. Sm. essential oil to the Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae). Journal of Insect Science. 10(162):1-11.
Wang, Y., Meng, Y., Zhai, C., Wang, M., Avula, B., Yuk, J., Smith, K.M., Isaac, G., Khan, I.A. 2019. The chemical characterization of Eleutherococcus senticosus and Ci-wu-jia Tea using UHPLC-UV-QTOF/MS. International Journal of Molecular Sciences. 20(3):475-488.
Avula, B., Bae, J., Chittiboyina, A.G., Wang, Y., Wang, M., Khan, I.A. 2019. Liquid chromatography-quadrupole time of flight mass spectrometric method for targeted analysis of 111 nitrogen-based compounds in weight loss and ergogenic supplements. Journal of Pharmaceutical and Biomedical Analysis. 174:305-323.
Yu, Q., Rao, R., Xu, X., Ganji, S., Jacob, M.R., Khan, S.I., Yu, B., Li, X. 2015. Antibacterial prenylated acylphloroglucinols from Psorothamnus fremontii. Organic Letters. 78:2748-2753.
Dasmahapatra, A.K., Khan, I.A. 2015. Modulation of DNA methylation machineries in Japanese rice fish (Oryzias latipes) embryogenesis by ethanol and 5-azacytidine. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 179:174-183.
Ali, A., Tabanca, N., Amin, E., Demirci, B., Khan, I.A. 2016. Chemical composition and biting deterrent activity of essential oil of Tagetes patula (Marigold) against Aedes aegypti. Journal of Medical Entomology. 11(10):1535-1538.
Chittiboyina, A.G., Avonto, C., Rua, D., Khan, I.A. 2015. Alternative testing methods for skin sensitization: NMR spectroscopy for probing the reactivity and classification of potential skin sensitizers. Chemical Research in Toxicology. 28:1704-1714.
Ravu, R.R., Jacob, M.R., Chen, X., Wang, M., Nasrin, S., Kloepper, J.W., Liles, M.R., Mead, D.A., Khan, I.A., Li, X. 2015. Bacillusin A, an antibacterial macrodiolide from Bacillus amyloliquefaciens. Journal of Natural Products. 78:924-928.
Wang, M., Zhao, J., Avula, B., Wang, Y., Avonto, C., Chittiboyina, A.G., Wylie, P.L., Parcher, J.F., Khan, I.A. 2014. High-resolution gas chromatography/mas spectrometry method for characterization and quantitative analysis of ginkgolic acids in ginkgo biloba plants, extracts, and dietary supplements. Journal of Agricultural and Food Chemistry. 62:12103-12111.
Avula, B., Sagi, S.J., Wang, Y., Wang, M., Garner, S., Manthey, J.A., Khan, I.A. 2016. Liquid chromatography-electrospray ionization mass spectrometry analysis of limonoids and flavanois in seeds of grapefruits, other citrus species, and dietary supplements. Planta Medica. 82:1058-1069.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. In a cooperative agreement with ARS researchers at Oxford, Mississippi, researchers at the National Center for Natural Products Research (NCNPR) at the University of Mississippi, Oxford, Mississippi, maintained basic discovery operations, with emphasis on the discovery of antifungals, cancer prevention agents, anti-inflammatory, anti-diabetic agents and immunomodulating agents. Continued to source plant materials for screening from our own plant collections and from numerous collaborators. We added 402 plant samples to our inventory this year and screened over 9, 801 natural product crude extracts, semi-purified fractions and purified compounds 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 immunomodulator/anti- inflammatory leads from natural sources, 110 compounds were isolated. In addition, 85 compounds were synthesized. Of the compounds tested, 16 were found to be biologically active for potential agricultural or medical uses. Many showed potent phytotoxic, antifungal, antibacterial, or antimalarial activities. In order to explore the antidiabetic potential of medicinal plants, about 50 plant extracts and 65 pure compounds were screened for PPAR agonistic activity. The actives were selected and were followed further in secondary assays such as adipogenic effect, antiadipogenic effect, and the agonistic effect towards LXR (which regulates lipid metabolism). 13 extracts were screened to find out if they possessed adipogenic effects (similar to rosiglitazone). A total of 22 (18 extracts and 4 pure compounds) were found to exhibit no adipogenic effects and they were selected to be tested for antiadipogenic effect to evaluate their potential against obesity and metabolic disorder as well as to understand their mechanism of antidiabetic and hypolipidemic action. Hypolipidemic action was determined in terms of activation of LXR pathway for 50 plant extracts and 64 pure compounds. 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 in field applications. About 150 plant extracts were screened for anti- inflammatory activities through target based cellular assays including NF- kB, iNOS and oxidative stress and over 140 pure compounds were screened for anti- inflammatory activities through these targets. The cytotoxicity of actives was also determined to find out the selectivity towards the target. About 10 extracts of medicinal plants and 25 isolated pure constituents from medicinal plants were also screened for their potential for causing drug interaction in terms of PXR activation and CYPs and P- glycoprotein inhibitions. Two plants were followed for in vivo anti diabetic efficacy in high fat diet fed animal model for diabetes, obesity and metabolic disorder. In collaboration with ElSohly Laboratories, Inc., continued development of a product shown to be effective in animal models for desensitization to poison ivy dermatitis. The lead compound in the poison ivy project has now been evaluated in a stage 1 clinical trial. 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. NCNPR finished construction in 2015 of a 96,000 sq. ft. research wing and occupancy of the new plant specimen repository is in progress. 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 01 Continued with development of agents for prevention/treatment of poison ivy dermatitis. Through a cooperative agreement with ARS researchers at Oxford, Mississippi, researchers at the National Center for Natural Products Research (NCNPR) at the University of Mississippi, 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 attained investigational new drug (IND) status by the U.S. Food and Drug Administration. A phase one trial has been completed by Hapten Sciences, Inc. Preparations are underway to initiate Phase II trial. Meanwhile, another formulation from one of our patented compounds prepared by Elsohly Lab, Inc. has been tested in guinea pigs at the center. This formulation was also found to be effective in preventing Poison Ivy dermatitis. 02 Identify dietary or botanical products with cancer chemopreventive potential. This program takes the most promising lead natural product extracts/pure compounds, identified using our battery of 13 cancer- related 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. During this period, ARS researchers at Oxford, Mississippi, have mostly focused on the mechanism of action of the most promising leads and have identified a potent inhibitor of glycolysis that when used in combination with a mitochondrial complex I inhibitor synergistically kills many types of human cancer cell lines and cells from patients tumors. We have also conducted mouse studies with this combination using the B16 melanoma model and have found significant inhibition of tumor development. 03 Continued development of immune-enhancing natural products and novel bioassays. Through a cooperative agreement with the ARS researchers at Oxford, Mississippi, researchers at the National Center for Natural Products Research (NCNPR) have developed a natural product extract from the cyanobacteria Arthrospira platensis that is enriched for Braun-type lipoproteins (potent toll-like receptor 2 agonists). Continued efforts focus on development of this product as a dietary-based approach for balancing innate immune function and its potential for enhancing the efficacy of immune checkpoint blockade in cancer therapy. A new project was also initiated with a company to help develop a mixture of natural products for treatment of bovine mastitis. During the last 15 years, NCNPR researchers have discovered that bacterial components (from endophytic bacteria) represent the major in vitro and in vivo innate immune cell activation agents within Echinacea and some other botanicals. This research received international recognition as the most innovative research published in Planta Medica during 2016. NCNPR researchers are also continuing to develop novel bioassays. In collaboration with Phytochemical Services Incorporated (Oxford, Mississippi) these researchers continue to offer and develop critically needed testing services for the standardization of immune-enhancing dietary supplements. These testing services are based on the novel concept of using bioassay-based standardization tools to quantitate the immune-enhancing potency within a broad range of supplements. In a separate project, a new bioassay system was set-up to discover natural products that expose beta glucans that are buried within the fungal cell wall, thus enabling the immune system to detect and elicit a response against these organisms. 04 New antifungal natural products for use in agriculture and medicine. Through a collaborative agreement ARS, researchers at Oxford, Mississippi and Agricen Sciences, with National Center for Natural Products Research, have identified a highly efficient microbial strain producing antifungal fusaricidins on a gram scale. A total of 48 fusaricidins including 28 new compounds have been characterized from this strain. The major compounds fusaricidins A and B account for 50% of the total fusaricidins. These compounds have demonstrated strong antifungal activities against a broad array of fungal pathogens including Cryptococcus neoformans. The structural diversity and potency of these fusaricidins makes this microbial strain as a potential bio- control agent for the treatment of fungal diseases in agricultural crops. The purified fusaricidins may serve as candidates for antifungal drug development. In a search for natural products that are potentiators of current antifungal drugs, mechanistic studies were performed. Three compounds were identified as potentiators of the cell wall damaging drug, caspofungin (CAS). The results revealed that a marine-derived cyclic peptide synergized with CAS by inducing the cell wall damage response pathway. These results were used to initiate a new target-based screen to identify compounds that induce this pathway. More than 2000 in-house compounds have been screened so far, and over 15 compounds were identified that strongly induced this pathway. Follow up studies are ongoing to determine if these compounds will synergize with CAS. NCNPR researchers also initiated a new assay to identify natural products that can function as B-glucan exposing agents, i.e. agents that can cause the B-glucans in the fungal cell wall to become exposed at the cell surface, thereby making the fungal cell visible to human immune cells. Once the optimization and validation is completed, several natural products will be screened to identify B-glucan exposing agents.

Impacts
(N/A)

Publications

Zhang, J., Zhao, J., Samoylenko, V., Jain, S., Tekwani, B.L., Muhammad, I. 2018. New polyisoprenylated polycyclic phloroglucines from Clusia gundlachii. Natural Product Communications. 13(3):361-365.
Parcher, J.F., Wang, M., Chittiboyina, A.G., Khan, I.A. 2018. In-source collision-induced dissociation (IS-CID): Applications, issues and structure elucidation with single-stage mass analyzers. Drug Testing and Analysis Journal. 10:28-36.
Wang, M., Haider, S., Chittiboyina, A.G., Parcher, J.F., Khan, I.A. 2018. 1,5-Dimethylhexylamine (octodrine) in sports and weight loss supplements: Natural constituent or synthetic chemical? Journal of Pharmaceutical and Biomedical Analysis. 152:298-305.
Zhao, J., Wang, M., Avula, B., Khan, I.A. 2018. Detection and quantification of phenethylamines in sports dietary supplements by NMR approach. Journal of Pharmaceutical and Biomedical Analysis. 151:347-355.
Raslan, A., Radwan, M., Ahmed, S., Nafady, A., Wanas, A., Babu, T., Hassanin, H., Elsohly, M. 2017. Evaluation of secondary metabolites from the Red Sea tunicate polyclinum constellatum. Pharmacy & Pharmacology International Journal. 5(3): doi:10.15406/ppij.2017.05.00123
Slater, S., Lasonkar, P.B., Haider, S., Alqahtani, M.J., Chittiboyina, A.G. , Khan, I.A. 2018. One-step, stereoselective synthesis of octahydrochromanes via the Prins reaction and their cannabinoid activities. Tetrahedron Letters. 59:807-810.
Wang, M., Raman, V., Zhao, J., Avula, B., Wang, Y., Wylie, P.L., Khan, I.A. 2018. Application of GC/Q-ToF combined with advanced data mining and chemometric tools in the characterization and quality control of bay leaves. Planta Medica.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. ARS Researchers at the Natural Products Utilization Research Unit (NPURU) at the University of Mississippi, Oxford, Mississippi, maintained basic discovery operations, with emphasis on the discovery of antifungals, anticancer, anti-inflammatory agents and immunomodulating agents. NPURU researchers continued to source plant materials for screening from our own plant collections and from numerous collaborators. We added 955 plant samples to our inventory this year and screened over 6700 natural product crude extracts, semi-purified fractions and purified compounds, 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 immunomodulator/anti-inflammatory leads from natural sources, 122 compounds were isolated. In addition, 45 compounds were synthesized and of the compounds tested, 21 were found to be biologically active for potential agricultural or medical uses. Many showed potent phytotoxic, antifungal, antibacterial, or antimalarial activities. In order to explore the antidiabetic potential of medicinal plants, about 150 plant extracts were screened for Peroxisome Proliferator- Activated Receptor (PPAR) agonistic activity. About 130 pure compounds were explored for antidiabetic potential. The actives 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. 52 extracts and 10 pure compounds were screened to find out if they possessed adipogenic effects (similar to rosiglitazone). A total of 29 (23 extracts and 6 pure compounds) were found to exhibit no adipogenic effects and they were selected to be tested for antiadipogenic effect to evaluate their potential against obesity and metabolic disorder as well as to understand their mechanism of antidiabetic and hypolipidemic action. 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 in field applications. About 200 plant extracts were screened for anti- inflammatory activities through target based cellular assays including Nuclear Factor kappa-light-chain-enhancer of activated B (NF-kB) cells, the inducible form of Nitric oxide synthase (iNOS), and oxidative stress, and over 100 pure compounds were screened for anti- inflammatory activities through these targets. About 50 extracts of medicinal plants and 35 isolated pure constituents from medicinal plants were also screened for their potential of causing drug interaction in terms of Pregnane X Receptor (PXR) activation and Cytochromes P450 (CYPs) and Permeability glycoprotein (P-gp) inhibitions. In collaboration with ElSohly Laboratories, Inc., continued development of a product shown to be effective in animal models for desensitization to poison ivy dermatitis. The lead compound in the poison ivy project is now being evaluated in a stage 1 clinical trial. 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 NPURU cultivates and processes medicinal plants to be used in the discovery program. Accomplishments 01 Continued with development of agents for prevention/treatment of poison ivy dermatitis. 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 attained investigational new drug (IND) status by the U.S. Food and Drug Administration and a phase 1 clinical trial is underway. 02 New animal model to evaluate natural product extracts for their potential as antidiabetic and anti-obesity products. This mouse model mimics type 2 diabetes in humans. ARS researchers in Oxford, Mississippi have tested two plant extracts (Thimelia hirsuta and Juniper communis). Both these extracts when given orally for a period of four weeks to mice; results showed promising hypoglycemic effect. A trend in lowering of plasma LDL-Cholesterol was also observed. Both the extracts were effective in controlling body weights of mice during the course of treatment. Experiments are underway to determine the minimum effective dose. ARS researchers in Oxford, Mississippi, have also set up a new animal model to study the effect of plant based immunomodulatory product(s) for effectiveness on minimizing the pathogenic effect of influenza virus infection. 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. 03 Identify dietary or botanical products with cancer chemopreventive potential. This program takes the most promising lead natural product extracts/pure compounds, identified using our battery of 13 cancer- related 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. During this period ARS researchers in Oxford, Mississippi, have identified 12 natural product extracts and several botanical combinations that substantially impact the growth of glioblastoma, triple-negative breast cancer and melanoma in vitro. 04 Continued development of immune enhancing natural products and bioassay methods for their standardization. ARS researchers in Oxford, Mississippi, have developed a natural product extract from the cyanobacteria Arthrospira platensis that is enriched for Braun-type lipoproteins (potent toll-like receptor 2 agonists). Animal studies are currently being conducted to evaluate the effectiveness of this cyanobacterial extract to be used as a dietary-based approach for balancing innate immune function that could result in more consistent patient responsiveness to immune checkpoint blockade in cancer therapy. NCNPR researchers are also continuing to collaborate with Phytochemical Services Incorporated, Oxford, Mississippi, to develop critically needed testing services and reference materials for the standardization of immune-enhancing dietary supplements. These supplements exhibit substantial variation in the levels of their active compounds and this has contributed to a lack of consistent product efficacy that has been observed in clinical studies. Two major hurdles have prevented the development of successful methods to standardize immune-enhancing dietary supplements so that these products can be manufactured to exhibit consistent efficacy. The first hurdle is the difficulty in identification of active compounds within each botanical. The second hurdle is the limitation of chemical standardization methods because the physiochemical properties measured by analytical techniques cannot predict the potency of the high molecular weight compounds that are responsible for the immune-enhancing effects of botanicals. NCNPR researchers have overcome both hurdles by translating our research discoveries on the active compounds in botanicals into bioassay-based standardization technology. Five in vitro bioassays have been developed that selectively quantitate different active compounds within a broad range of immune supplements. 05 New antifungal natural products for use in agriculture and medicine. ARS researchers in Oxford, Mississippi, have continued work on identifying new antifungal compounds that can potentiate the activity of current antifungal drugs, especially for the drug-resistant fungal pathogens. Activities in this program also include determining their mechanism of action (MOA) and evaluating their in vivo efficacy. ARS researchers in Oxford, Mississippi, evaluated three compounds with Ribonucleic acid sequencing (RNA-Seq) to identify the potential pathways. Two of the compounds targeted pathways not known to be targeted by current drugs. These included a plant-derived acylphloroglucinol analog and a microbial-derived cyclic peptide. RNA- Seq results revealed that the former targets mitochondrial functions and the latter targets calcium homeostasis. The third compound evaluated was a plant-derived steroidal alkaloid, and its RNA-Seq profile revealed that it targets the fungal cell wall. It is worth noting that the clinically used echinocandin antifungal drugs also target the cell wall; however, they are inactive against Cryptococcus (C.) neoformans, one of the major fungal pathogens. Thus, given that cell wall-targeting drugs have fungal specificity, this work has the potential to lead to the development of a novel anti-cryptococcal drug with high specificity and low toxicity. This year, 2 compounds were evaluated to verify their MOA. Previous results revealed that these compounds target a pathway associated with the synthesis of iron-sulfur clusters in fungal cells. Both compounds specifically inhibited the activities of enzymes containing iron-sulfur clusters without affecting the activities of non-iron-sulfur enzymes. This year, mechanistic studies were also conducted on 3 compounds that potentiate the activity of the antifungal drug caspofungin and follow up studies are ongoing.

Impacts
(N/A)

Publications

Tyler, H.L., Haron, M.H., Pugh, N.D., Zhang, J., Jackson, C.R., Pasco, D.S. 2016. Bacterial components are the major contributors to the macrophage stimulating activity exhibited by extracts of common edible mushrooms. Food & Function. 7:4213-4221.
Avula, B., Wang, Y., Isaac, G., Yuk, J., Wrona, M., Yu, K., Khan, I.A. 2017. Metabolic profiling of Hoodia, Chamomile, Terminalia Species and evaluation of commercial preparations using Ultra-High Performance Quadrupole Time of Flight-Mass Spectrometry. Planta Medica. doi:10.1055/s- 0043-109239.
Zulfiqar, F., Khan, S.I., Ross, S.A., Ali, Z., Khan, I.A. 2017. Prenylated flavonol glycosides Epimedium grandiflorum: Cytotoxicity and evaluation against inflammation and metabolic disorders. Phytochemistry Letters. 20:160-167.
Raman, V., Tabanca, N., Demirci, B., Khan, I.A. 2017. Studies on the floral anatomy and scent chemistry of Titan Arum (Amorphophallus titanum, Araceae). Turkish Journal of Botany. 41:63-74.
Hu, S., Wang, Y., Avula, B., Wang, M., Khan, I.A. 2017. Separation of cucurbitane triterpenoids from bitter melon drinks and determination of partition coefficients using vortex-assisted dispersive liquid-phase microextraction followed by UHPLC analysis. Journal of Separation Science. 40(10):2238-2245.
Gross, A., Tabanca, N., Islam, R., Ali, A., Khan, I.A., Kaplancikli, Z.A., Altintop, M.D., Ozdemir, A., Bloomquist, J.R. 2017. Toxicity and synergistic activities of Chalcones against Aedes aegypti (Diptera: Culicidae) and Drosophila melanogaster (Diptera: Drosophilidae). Journal of Medical Entomology. 54(2):382-386.
Seo, E., Wu, C., Ali, Z., Wang, Y., Khan, S.I., Walker, L.A., Khan, I.A., Efferth, T. 2016. Both phenolic and non-phenolic green tea fractions inhibit migration of cancer cells. Frontiers in Pharmacology. 7(398):1-16.
Yalamanchili, C., Manda, V.K., Chittiboyina, A.G., Guernieri, R.L., Harrell, Jr., W.A., Webb, R.P., Smith, L.A., Khan, I.A. 2016. Utilizing Ayurvedic literature for the identification of novel phytochemical inhibitors of botulinum neurotoxin A. Journal of Ethnopharmacology. 197:211-217.
Gil, G., Mao, P., Avula, B., Ali, Z., Chittiboyina, A.G., Khan, I.A., Walker, L.A., Wang, D. 2017. Proteoform-specific protein binding of small molecules in complex matrices. ACS Chemical Biology. 12:389-397.
Chittiboyina, A.G., Avonto, C., Khan, I.A. 2016. What happens after the activation of ascaridole? Reactive compounds and their implications for skin sensitization. Chemical Research in Toxicology. 29:1488-1492. doi:10. 1021/acs.chemrestox.6b00157.
Yu, Q., Ravu, R., Jacob, M.R., Khan, S.I., Agarwal, A.K., Yu, B., Li, X. 2016. Synthesis of natural acylphloroglucinol-based antifungal compounds against Cryptococcus species. Journal of Natural Products. 79:2195-2201. doi:10.1021/acs.jnatprod.6b00224.
Shukla, S., Shariat-Madar, Z., Walker, L.A., Tekwani, B.L. 2016. Mechanism for neurotropic action of vorinostat, a pan histone deacetylase inhibitor. Molecular and Cellular Neuroscience. 77:11-20.
Ali, A., Cantrell, C.L., Khan, I. 2017. A new in vitro bioassay system for discovery and quantitative evaluation of mosquito repellents. Journal of Medical Entomology. 54(5):1328-1336. doi:10.1093/jme/tjx100.
Zhai, C., Wang, M., Raman, V., Rehman, J.U., Meng, Y., Zhao, J., Avula, B., Wang, Y., Tian, Z., Khan, I.A. 2016. Eleutherococcus senticosus (Araliaceae) leaf morpho-anatomy, essential oil composition and its biological activity against Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology. 54(3):658-669. doi:10.1093/jme/tjw221.
Jain, J., Jain, S., Walker, L., Tekwani, B. 2017. Inhibitors of ubiquitin E3 ligase as potential new antimalarial drug leads. Biomed Central Pharmacology and Toxicology Journal. doi:10.1186/s40360-017-0147-4.
Manda, V.K., Avula, B., Ashfaq, K., Abe, N., Khan, I.A., Khan, S.I. 2016. Quantification of mesembrine and mesembrenone in mouse plasma using UHPLC- QToF-MS: Application to a pharmacokinetic study. Biomedical Chromatography. doi:10.1002/bmc.3815.
Aguirre, L., Milton-Laskibar, I., Hijona, E., Bujanda, L., Rimando, A.M., Poetillo, M.P. 2017. Effects of pterostilbene in brown adipose tissue from obese rats. Journal of Physiology and Biochemistry. 73(3):457-464. doi:10. 1007/s13105-017-0556-2.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. Researchers at the National Center for Natural Products Research (NCNPR) at the University of MS, Oxford, MS maintained basic discovery operations, with emphasis on the discovery of antifungals, anticancer, anti- inflammatory agents and immunomodulating agents. Continued to source plant materials for screening from our own plant collections and from numerous collaborators. Added 1057 plant samples to our inventory this year. Screened over 20,000 natural product crude extracts, semi-purified fractions and purified compounds for biological activities against specific molecular targets and whole cell systems. As part of our continuing effort in the search for anti-infective, anticancer, and immunomodulator/anti-inflammatory leads from natural sources, 128 compounds were isolated, 62 of which are novel. In addition, 53 compounds related to these isolates were synthesized. Of the compounds tested, 36 were found to be biologically active for potential agricultural or medical uses. Many showed potent phytotoxic, antifungal, antibacterial, or antimalarial activities. Approximately 250 of our isolated actives or extracts have been characterized in more detailed follow-up assays to determine their mode of action, pharmaceutical properties, toxicity, and selectivity across a range of assays. 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 in field applications. About 100 plant extracts were screened for anti-inflammatory activities through target based cellular assays including NF-kB, iNOS and oxidative stress and over 100 pure compounds were screened for anti-inflammatory activities through these targets. About 100 plant extracts and 100 pure compounds were screened for anticancer properties against human tumor cell lines. About 50 plant extracts were screened for PPAR activation activity in order to explore their antidiabetic potential. About 80 pure compounds were explored for antidiabetic potential. The actives were selected and were followed further in secondary assays such as western blots and RT-PCR to look into the expression of target proteins and evaluate the mechanism of action. About 25 extracts of medicinal plants and 40 isolated constituents from medicinal plants were also screened for their potential of causing drug interaction in terms of PXR activation and CYPs and P-gp inhibitions. Several were followed in secondary assays of enzymatic activity and gene expression analysis. In collaboration with ElSohly Laboratories, Inc., continued development of a product shown to be effective in animal models for desensitization to poison ivy dermatitis. The lead compound in the poison ivy project is now being evaluated in a stage 1 clinical trial. 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 are being developed. Here NCNPR cultivates and processes medicinal plants to be used in the discovery program. NCNPR finished construction in 2015 of a 96,000 sq. ft. research wing and final stages of occupancy are in progress. 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 01 Continued with development of agents for prevention/treatment of poison ivy dermatitis. Scientists at the NCNPR at the University of 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 attained investigational new drug (IND) status by the US Food and Drug Administration and a phase 1 clinical trial is underway. 02 New scientific technologies that improve laboratory operations. Scientists at the NCNPR invented a chromatographic device which uses centrifugal force to efficiently separate complex mixtures of organic compounds into their various components. A prototype instrument was built and tested. A patent application and research disclosure have been filed. An option agreement has been executed with a company for commercialization of the product. This will provide new solutions to chromatographic challenges faced by scientists and laboratory technicians in many fields, including agriculture, pharmaceutics, and food science. 03 Develop treatments for cancer. A cancer research program requires a drug discovery program in order to explore all avenues of treatment. The Cancer Drug Discovery Core of the University of Mississippi Cancer Institute is a cooperative venture of the School of Pharmacy and the Medical Center. This program takes the most promising lead natural product extracts/pure compounds, identified using our battery of 13 cancer-related 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. A new faculty member added this year will oversee translation of natural product leads identified by the Cancer Drug Discovery Core by characterizing those that enhance responsiveness of various tumor types to chemotherapy using a chemosensitivity testing approach (ChemoID�) developed and patented by him. During this period, we have been transitioning this assay to a 384 well format and focusing on identification of natural product extracts and pure compounds that impact glioblastoma in vitro. Currently a trial of 100 patients with glioblastoma is underway using ChemoID�; these patient derived tumor cells in culture will be used to evaluate our natural product leads. This represents an approach that could afford us an important and unique niche in the Precision Medicine Initiative - identifying patient/ tumor-specific natural product-chemotherapeutic combinations that are effective against both bulk tumor cells and tumor stem-like cells. 04 Discovery of new drugs to overcome resistance in bacteria. Bacterial resistance to currently used antibiotics has become a worldwide health concern, in particular drug resistance has increased dramatically in the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter). We have developed a primary screen to evaluate the ability of natural products to reverse drug resistance. Resistant organisms are treated with natural products in the presence of sub- inhibitory concentrations of antibiotics. These organisms are resistant because they produce ESBL, NDM and KPC beta lactamases the major causes of clinical resistance and antibiotic failure. Continued screening of our natural product samples in these assays is underway. 05 Continued development of immune enhancing natural products and bioassay methods for their standardization. Scientists at the NCNPR have developed a natural product extract from the cyanobacteria Arthrospira platensis that contains a very potent Toll-like receptor 2 (TLR2) agonist. Published research supports the role of TLR2 agonists in maintaining intestinal epithelial barrier function. Increased permeability of the intestinal epithelium is associated with increased inflammation, depression and other pathophysiological conditions. We are establishing collaborations with scientists at The University of Mississippi Medical Center to perform clinical trials on the cyanobacterial extract for use in preventing cancer therapy-induced GI mucositis and treatment of depression. We are also working with the Department of Health, Exercise Science, and Recreation Management at The University of Mississippi to plan a clinical trial evaluating the effectiveness of the cyanobacterial extract in prevention of delayed onset muscle soreness. In collaboration with Phytochemical Services Incorporated (Oxford, MS) we have set-up a battery of different in vitro bioassay methods capable of quantitating the potency of natural product materials that enhance immune function. These bioassays are expected to overcome the limitations of chemical standardization methods since physiochemical properties cannot fully predict the biological potency of the high molecular weight substances that are typically responsible for the immune enhancing effects of natural product materials. Bioassay standardization methods provide the means to fully characterize immune enhancing natural product materials for use in research, clinical trials and the consumer market. 06 New antifungal natural products for use in agriculture and medicine. Scientists at the NCNPR are working on discovering novel antifungal compounds for treating life-threatening opportunistic fungal infections. In addition, projects are ongoing for discovering compounds that can potentiate the activity of current antifungal drugs, especially in drug- resistant fungal pathogens. This year, scientists in this program evaluated the mechanism behind the synergistic effects of two different natural products in combination with the antifungal drugs amphotericin B and caspofungin. Transcript profiling analysis revealed that these compounds prevented fungal cells from adapting to the specific stress exerted by each drug. In addition, RNA-Seq technology was used to determine that each compound targeted a unique cellular pathway that was different from that targeted by the antifungal drug. These discoveries will allow the development of new therapies that will improve the activity of current antifungal drugs in pathogens that have become resistant to them. This year, the amphotericin B-potentiating compound was advanced to an in vivo evaluation study in a mouse model of fungal infection in our collaborator�s lab at Duke University. Finally, this year, in addition to the analysis of in-house compounds, studies were also conducted on commercially available collections of drug-like molecules. Using a molecular assay, ~12,000 molecules were evaluated. Eight drug-like molecules were identified that potentiated the activity of the antifungal drug caspofungin in fungal pathogens, especially in drug-resistant strains. Further studies are ongoing to evaluate the in vivo efficacy of these compounds in combination with caspofungin and to characterize their drug-potentiating mechanism. These accomplishments may lead to new treatments for numerous diseases of plants, animals, and humans.

Impacts
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Publications

Wang, Y., Avula, B., Abe, N., Wei, F., Wang, M., Ma, S., Ali, Z., Elsohly, M.A., Khan, I.A. 2016. Tandem Mass Spectrometry for Structural Identification of Sesquiterpene alkaloids from the stems of dendrobium nobile using LC-QToF3. Planta Medica. 82(7):662-70.
Haron, M.H., Tyler, H.L., Pugh, N.D., Moraes, R.M., Maddox, V.L., Jackson, C.R., Pasco, D.S. 2016. Activities and prevalence of proteobacteria members colonizing Echinacea purpurea fully account for in vitro macrophage activation exhibited by extracts of this botanical. Planta Medica. 89(4-5):451-62. DOI: 10.1007/s11103-015-0381-3
Jain, S., Jacob, M., Walker, L., Tekwani, B. 2016. Screening North American plant extracts in vitro against Trypanosoma brucei, the causative agent for Human African Trypanosomiasis. BMC Complementary and Alternative Medicine. 16(1):131. DOI: 10.1186/s12906-016-1122-0.
Bufalo, J., Cantrell, C.L., Jacob, M.R., Schrader, K., Tekwani, B.L., Kustova, T.S., Ali, A., Boaro, C.S. 2015. Antimicrobial and antileishmanial activities of diterpenoids isolated from the roots of Salvia deserta. Planta Medica. 82:131-137.
Nicolau-Goncalves, V., Cantrell, C.L., Wedge, D.E., Ferreira, M.C., Soares, M.A., Jacob, M.R., Oliveira, F., Galante, D., Rodrigues, F., Alves, T., Zani, C., Junior, P., Murta, S., Romanho, A., Barbosa, E., Kroon, E., Oliveira, J., Gomez-Silva, B., Galetovic, A., Rosa, C.A., Rosa, L. 2015. Fungi associated with rocks of the Atacama Desert: taxonomy, distribution, diversity, ecology and bioprospection for bioactive compounds. Environmental Microbiology. 18:232-245.
Avula, B., Chittiboyina, A.G., Wang, Y., Sagi, S.J., Raman, V., Wang, M., Khan, I.A. 2016. Simultaneous determination of aegeline and six coumarins from different parts of the plant Aegle marmelos using UHPLC-PDA-MS and chiral separation of aegeline using HPLC-ToF-MS . Planta Medica. 82(06) :580-588.
Meepagala, K.M., Johnson, R.D., Techen, N., Wedge, D.E., Duke, S.O. 2015. Phomalactone from a phytopathogenic fungus infecting Zinnia elegans (Asteraceae) leaves. Journal of Chemical Ecology. 41:602-612.
Wang, M., Carrell, E., Chittiboyina, A.G., Avula, B., Wang, Y., Zhao, J., Parcher, J.F., Khan, I.A. 2016. Concurrent and supercritical fluid chromatographic analysis of Terpene Lactones and ginkolic acids in Ginko biloba. Analytical and Bioanalytical Chemistry. 408(17):4649-4660. DOI: 10. 1007/s00216-016-9544-6.
Zhang, Z., Zulfiqar, A., Khan, S.I., Khan, I.A. 2016. Cytotoxic monacolins from red yeast rice, a Chinese medicine and food. Journal of Natural Products. 202:262-8. DOI:10.1016/j.foodchem.2015.12.039.
Aumsuawan, P., Khan, S.I., Khan, I.A., Ali, Z., Avula, B., Walker, L.A., Shariat-Madar, Z., Helferich, W.G., Katzenellenbogen, B.S., Dasmahapatra, A.K. 2016. The anticancer potential of steroidal saponin, dioscin, isolated from wild yam (Dioscorea villosa)root extract in invasive human breast cancer cell line MDA-MB-231 in vitro. Archives Of Biochemistry and Biophysics. 591:98-110. DOI: 10.1016/j.abb.2015.12.001.
Manda, V.K., Ibrahim, M.A., Dale, O.R., Kumarihamy, M., Cutler, S.J., Khan, I.A., Walker, L.A., Muhammad, I., Khan, S.I. 2016. Modulation of CYPs, P- gp, and PXR by Eschscholzia californica (California poppy) and its alkaloids. Planta Medica. 82:551-558. DOI
Lata, H., Chandra, S., Wang, Y., Elsohly, M.A., Khan, I.A. 2015. Polyhouse cultivation of invitro raised elite Stevia rebaudiana Bertoni: An assessment of biochemical and photosynthetic characteristics. Journal of Tropical Agriculture. 33(4):1-7. 2015
Dasmahapatra, A.K., Khan, I.A. 2015. DNA methyltransferase expressions in Japanese rice fish (Oryzias latipes) embryogenesis is developmentally regulated and modulated by ethanol and 5-azacytidine. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 13:176-177 (2015).
Aumsuwan, P., Khan, S.I., Khan, I.A., Walker, L.A., Dasmahapatra, A.K. 2016. Gene expression profiling and pathway analysis in MCF-7 and MDA-MB- 231 human breast cancer cell lines treated with dioscin. Data in Brief. (2016)272-279.
Avula, B., Sagi, S.J., Gafner, S., Upton, R., Wang, Y., Wang, M., Khan, I. A. 2015. Identification of Ginkgo biloba supplements adulteration using HPTLC and UHPLC-DAD-QToF-MS. Analytical and Bioanalytical Chemistry. 407(25):7733-7746. DOI 10.1007/s00216-015-8938-1.
Kaymakcioglu, B.K., Beyhan, N., Tabanca, N., Ali, A., Wedge, D.E., Duke, S. O., Bernier, U.R., Khan, I.A. 2015. Discovery and structure activity relationships of 2-pyrazolines derived from chalcones from a pest management perspective. Journal of Agricultural and Food Chemistry. 24:3632-3644.
Parveen, I., Wang, M., Zhao, J., Chittiboyina, A.G., Tabanca, N., Ali, A., Baerson, S.R., Techen, N., Chappell, J., Khan, I.A., Pan, Z. 2015. Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and a-bisabolene synthases. Plant Molecular Biology. 89:451-462.
Ding, Y., Liu, H., Tekwani, B.L., Nanayakkara, N., Khan, I.A., Walker, L.A. , Doerksen, R.J. 2016. Methemoglobinemia hemotoxicity of some antimalarial 8-aminoquinoline analogues and their hydroxylated derivatives: density functional theory computation of ionization potentials. Chemical Research in Toxicology.
Li, C., Ren, G., Yang, B., Miklossy, G., Turkson, J., Fei, P., Ding, Y., Walker, L.A., Cao, S. 2016. Meroterpenoids with antiproliferative activity from a Hawaiian-plant associated fungus Peyronellaea coffeae-arabicae FT238. Organic Letters. 18(10):2335-8. doi:10.1021/acs.orglett.6b00685.
Li, C., Ding, Y., Yang, B., Miklossy, G., Yin, H., Walker, L.A., Turkson, J., Cao, S. 2015. A New Metabolite with a unique 4-pyranone-ylactam-1,4- thiazine moiety from a Hawaiian-plant associated fungus. Organic Letters. 17(14):3556-9. doi:10.1021/acs.orglett.5b01650.
Tekwani, B.L., Avula, B., Sahu, R., Chaurasiya, N.D., Khan, S.I., Jain, S., Fasinu, P.S., Herath, H.B., Stanford, D., Nanayakkara, N.D., Mcchesney, J. D., Yates, T.W., Elsohly, M.A., Khan, I.A., Walker, L.A. 2015. Enantioselective pharmacokinetics of primaquine in healthy human volunteers. Drug Metabolism and Disposition. 43(4):571-7.
Avonto, C., Chttiboyina, A.G., Rua, D., Khan, I.A. 2015. A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers. Journal of Toxicology and Applied Pharmacology. 289:177-184.
Cimmino, A., Evidente, M., Masi, M., Ali, A., Tabanca, N., Khan, I.A., Evidente, A. 2015. Papyracillic acid and its derivatives as biting deterrents against Aedes aegypti(Diptera: Culicidae): structure�activity relationships. Medical Chemistry Research. 24:3981-3989.
Avonto, C., Chittiboyina, A.G., Wang, M., Vasquez, Y., Rua, D., Khan, I.A. 2016. Sensitation potential of tea tree essential oils. Impact of the chemical composition on aging and generation of reactive species. Chemical Research in Toxicology. DOI: 10.1021/acs.chemrestox.5b00530.
Li, C., Ding, Y., Yang, B., Hoffman, N., Yin, H., Mahmud, T., Turkson, J., Cao, S. 2016. Ermophilane sesquiterpenes from Hawaiian endophytic fungus Chaetoconis sp.FT087. Phytochemistry. 126:41-6. doi:doi:10.1016/j. phytochem.2016.03.005.
Mohamed, S.M., Bachkeet, E.Y., Bayoumi, S.A., Jain, S., Cutler, S., Tekwani, B.L., Ross, S.A. 2015. Potent antitrypanosomal triterpenoid saponins from Mussaenda luteola. Fitoterapia. 107:114-121.
Radwan, M., Wanas, A.S., Fronczek, F.R., Jacob, M.R., Ross, S.A. 2015. Polybrominated diphenyl ethers from the marine organisms Lendenfeldia dendyi and Sinularia dura with anti-MRSa activity. Medical Chemistry Research. 24:3398-3404.
Gorovoi, P.G., Suleimen, E.M., Dudkin, R.V., Wang, M., Khan, I.A., Ross, S. A. 2015. Constituent composition and biological activity of Nepeta manchuriensis essential oil. Chemistry of Natural Compounds. 51(5):989-990.
Fasinu, P.S., Gurley, B.J., Walker, L.A. 2015. Clinically relevant pharmacokinetic herb-drug interactions in antiretroviral therapy. Current Drug Metabolism. 17(1):52-64.