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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

2017 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
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.

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

Review 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.