Carrie Waterman

Anthelmintic screening of Sub-Saharan African plants used in traditional medicine

AIM OF STUDY This study screened for anthelmintic activity of plant species traditionally used in the treatment of intestinal parasites and their symptoms in Sub-Saharan Africa in an effort to confirm their local use and aid in the search for new compounds since resistance is a growing concern. MATERIALS AND METHODS Aqueous and organic extracts of 33 plant parts from 17 plant species traditionally used in the treatment of intestinal infections in Sub-Saharan Africa were evaluated for their anthelmintic activity. This activity was assessed using a standard motility assay against a levamisole resistant strain of the nematode Caenorhabditis elegans. RESULTS AND CONCLUSIONS Anthelmintic activity was confirmed in 12 plant species. Of these, eight showed strong evidence of activity (p<0.0001), one exhibited moderate evidence of activity (p<0.001), three demonstrated weak evidence of activity (p<0.05), and five plants showed no evidence of activity. The eight species with the strongest evidence of activity were Acacia polyacantha, Anogeissus leiocarpus, Bridelia micrantha, Cassia sieberiana, Combretum nigricans, Grewia bicolor, Strychnos spinosa and Ziziphus mucronata. In only two cases, Anogeissus leiocarpus and Cassia sieberiana, anthelmintic activity has been previously confirmed. The activity demonstrated against the levamisole resistant strain of Caenorhabditis elegans and the presence of molecules in these plants known or suspected of having a broad spectrum of activity provide support for further study of these plants and their compounds as possible treatments for parasitic worm infections.

Isothiocyanate-rich Moringa oleifera extract reduces weight gain, insulin resistance, and hepatic gluconeogenesis in mice.

SCOPE Moringa oleifera (moringa) is tropical plant traditionally used as an antidiabetic food. It produces structurally unique and chemically stable moringa isothiocyanates (MICs) that were evaluated for their therapeutic use in vivo. METHODS AND RESULTS C57BL/6L mice fed very high fat diet (VHFD) supplemented with 5% moringa concentrate (MC, delivering 66 mg/kg/d of MICs) accumulated fat mass, had improved glucose tolerance and insulin signaling, and did not develop fatty liver disease compared to VHFD-fed mice. MC-fed group also had reduced plasma insulin, leptin, resistin, cholesterol, IL-1β, TNFα, and lower hepatic glucose-6-phosphatase (G6P) expression. In hepatoma cells, MC and MICs at low micromolar concentrations inhibited gluconeogenesis and G6P expression. MICs and MC effects on lipolysis in vitro and on thermogenic and lipolytic genes in adipose tissue in vivo argued these are not likely primary targets for the anti-obesity and anti-diabetic effects observed. CONCLUSION Data suggest that MICs are the main anti-obesity and anti-diabetic bioactives of MC, and that they exert their effects by inhibiting rate-limiting steps in liver gluconeogenesis resulting in direct or indirect increase in insulin signaling and sensitivity. These conclusions suggest that MC may be an effective dietary food for the prevention and treatment of obesity and type 2 diabetes.

Screening Mangrove Endophytic Fungi for Antimalarial Natural Products

We conducted a screening campaign to investigate fungi as a source for new antimalarial compounds. A subset of our fungal collection comprising Chinese mangrove endophytes provided over 5000 lipophilic extracts. We developed an accelerated discovery program based on small-scale cultivation for crude extract screening and a high-throughput malaria assay. Criteria for hits were developed and high priority hits were subjected to scale-up cultivation. Extracts from large scale cultivation were fractionated and these fractions subjected to both in vitro malaria and cytotoxicity screening. Criteria for advancing fractions to purification were developed, including the introduction of a selectivity index and by dereplication of known metabolites. From the Chinese mangrove endophytes, four new compounds (14–16, 18) were isolated including a new dimeric tetrahydroxanthone, dicerandrol D (14), which was found to display the most favorable bioactivity profile.

Comparison of motility, recovery, and methyl-thiazolyl-tetrazolium reduction assays for use in screening plant products for anthelmintic activity

The primary objective of these experiments was to compare the effectiveness of motility, recovery, and methyl-thiazolyl-tetrazolium (MTT) reduction assays for determining anthelmintic activity of plant extracts and purified compounds from these extracts. Caenorhabditis elegans was used as the test organism. High-performance liquid chromatography (HPLC) grade water and M9 medium were used as the solvents. Copper, a common metal pollutant, and the anthelmintic drug levamisole were used as reference compounds. Extracts from the West African plant Anogeissus leiocarpus, which is used to treat worm infections, as well as two active compounds found in this plant, gallic and gentisic acids, were included in this comparison. MTT assay results for viability of worms were significantly lower (p < 0.01) than motility and recovery assay results. However, both gallic acid and the plant extract, in the absence of worms, caused reduction of MTT. Worm survival for levamisole using M9 medium was significantly (p < 0.01) higher than for HPLC grade water for all three methods. On the other hand, gallic acid showed significant (p < 0.05) activity in M9 medium but no activity in HPLC grade water, whereas gentisic acid was effective in HPLC grade water but had no activity in M9 medium. Activity of the A. leiocarpus extract also varied with solvent. In conclusion, plant extracts can be screened using motility assays that include both HPLC grade water and M9 salts.

Miniaturized Cultivation of Microbiota for Antimalarial Drug Discovery.

The ongoing search for effective antiplasmodial agents remains essential in the fight against malaria worldwide. Emerging parasitic drug resistance places an urgent need to explore chemotherapies with novel structures and mechanisms of action. Natural products have historically provided effective antimalarial drug scaffolds. In an effort to search natures chemical potential for antiplasmodial agents, unconventionally sourced organisms coupled with innovative cultivation techniques were utilized. Approximately 60,000 niche microbes from various habitats (slow‐growing terrestrial fungi, Antarctic microbes, and mangrove endophytes) were cultivated on a small‐scale, extracted, and used in high‐throughput screening to determine antimalarial activity. About 1% of crude extracts were considered active and 6% partially active (≥67% inhibition at 5 and 50 μg/mL, respectively). Active extracts (685) were cultivated on a large‐scale, fractionated, and screened for both antimalarial activity and cytotoxicity. High interest fractions (397) with an IC50 < 1.11 μg/mL were identified and subjected to chromatographic separation for compound characterization and dereplication. Identifying active compounds with nanomolar antimalarial activity coupled with a selectivity index tenfold higher was accomplished with two of the 52 compounds isolated. This microscale, high‐throughput screening project for antiplasmodial agents is discussed in the context of current natural product drug discovery efforts.

Introducing experimental design by evaluating efficacy of herbal remedies (Do herbal remedies really work

This paper is based upon experiments developed as part of a Directed Research course designed to provide undergraduate biology students experience in the principles and processes of the scientific method used in biological research. The project involved the evaluation of herbal remedies used in many parts of the world in the treatment of diseases producing diarrhea as a major symptom. Methods used for testing the efficacy of these remedies vary greatly, and this provides an opportunity for inquiry in the classroom. The nematode Caenorhabditis elegans is used as the test organism. Survival of this worm is easily determined by assessing motility using a dissection microscope. The influence of two solvents commonly used for testing these treatments, M9 salt solution and purified water, on survival of worms is examined. The results were important to a graduate project evaluating the influence of these solvents on bioassay sensitivity testing partially purified extracts of the West African plant, Anogeissus leiocarpus, used for treatment of diarrhea. Directed research projects allow undergraduate biology students to become engaged in science and develop a deeper understanding of science process skills. The experiments can be extended to examine other variables as directed research projects or modified to use as experimental case examples as part of a laboratory exercise.