RESUMO
Although tannin-rich forages are known to increase protein uptake and to reduce gastrointestinal nematode infections in grazing ruminants, most published research involves forages with condensed tannins (CT), while published literature lacks information on the anthelmintic capacity, nutritional benefits, and antioxidant capacity of alternative forages containing hydrolyzable tannins (HT). We evaluated the anthelmintic activity and the antioxidant capacity of plant extracts containing either mostly CT, mostly HT, or both CT and HT. Extracts were prepared with 70% acetone, lyophilized, redissolved to doses ranging from 1.0mg/mL to 25mg/mL, and tested against adult Caenorhabditis elegans as a test model. The extract concentrations that killed 50% (LC(50)) or 90% (LC(90)) of the nematodes in 24h were determined and compared to the veterinary anthelmintic levamisole (8 mg/mL). Extracts were quantified for CT by the acid butanol assay, for HT (based on gallic acid and ellagic acid) by high-performance liquid chromatography (HPLC) and total phenolics, and for their antioxidant activity by the oxygen radical absorbance capacity (ORAC) assay. Extracts with mostly CT were Lespedeza cuneata, Salix X sepulcralis, and Robinia pseudoacacia. Extracts rich in HT were Acer rubrum, Rosa multiflora, and Quercus alba, while Rhus typhina had both HT and CT. The extracts with the lowest LC(50) and LC(90) concentrations, respectively, in the C. elegans assay were Q. alba (0.75 and 1.06 mg/mL), R. typhina collected in 2007 (0.65 and 2.74 mg/mL), A. rubrum (1.03 and 5.54 mg/mL), and R. multiflora (2.14 and 8.70 mg/mL). At the doses of 20 and 25mg/mL, HT-rich, or both CT- and HT-rich, extracts were significantly more lethal to adult C. elegans than extracts containing only CT. All extracts were high in antioxidant capacity, with ORAC values ranging from 1800 µmoles to 4651 µmoles of trolox equivalents/g, but ORAC did not correlate with anthelmintic activity. The total phenolics test had a positive and highly significant (r=0.826, p ≤ 0.01) correlation with total hydrolyzable tannins. Plants used in this research are naturalized to the Appalachian edaphoclimatic conditions, but occur in temperate climate areas worldwide. They represent a rich, renewable, and unexplored source of tannins and antioxidants for grazing ruminants, whereas conventional CT-rich forages, such as L. cuneata, may be hard to establish and adapt to areas with temperate climate. Due to their high in vitro anthelmintic activity, antioxidant capacity, and their adaptability to non-arable lands, Q. alba, R. typhina, A. rubrum, and R. multiflora have a high potential to improve the health of grazing animals and must have their anthelmintic effects confirmed in vivo in both sheep and goats.
Assuntos
Anti-Helmínticos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Taninos Hidrolisáveis/análise , Extratos Vegetais/farmacologia , Proantocianidinas/análise , Árvores/química , Acer/química , Animais , Anti-Helmínticos/química , Anti-Helmínticos/isolamento & purificação , Antioxidantes/análise , Fagaceae/química , Fenóis/análise , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Caules de Planta/química , Rhus/química , Robinia/química , Salix/químicaRESUMO
The most challenging obstacles to testing products for their anthelmintic activity are: (1) establishing a suitable nematode in vitro assay that can evaluate potential product use against a parasitic nematode of interest and (2) preparation of extracts that can be redissolved in solvents that are miscible in the test medium and are at concentrations well tolerated by the nematode system used for screening. The use of parasitic nematodes as a screening system is hindered by the difficulty of keeping them alive for long periods outside their host and by the need to keep infected animals as sources of eggs or adults when needed. This method uses the free-living soil nematode Caenorhabditis elegans as a system to screen products for their potential anthelmintic effect against small ruminant gastrointestinal nematodes, including Haemonchus contortus. This modified method uses only liquid axenic medium, instead of agar plates inoculated with Escherichia coli, and two selective sieves to obtain adult nematodes. During screening, the use of either balanced salt solution (M-9) or distilled water resulted in averages of 99.7 (± 0.73)% and 96.36 (± 2.37)% motile adults, respectively. Adult worms tolerated DMSO, ethanol, methanol, and Tween 80 at 1% and 2%, while Labrasol (a bioenhancer with low toxicity to mammals) and Tween 20 were toxic to C. elegans at 1% and were avoided as solvents. The high availability, ease of culture, and rapid proliferation of C. elegans make it a useful screening system to test plant extracts and other phytochemical compounds to investigate their potential anthelmintic activity against parasitic nematodes.
Assuntos
Anti-Helmínticos/farmacologia , Extratos Vegetais/farmacologia , Drogas Veterinárias/farmacologia , Animais , Anti-Helmínticos/química , Caenorhabditis elegans , Extratos Vegetais/química , Solventes , Drogas Veterinárias/químicaRESUMO
There is a pressing need to develop alternative, natural anthelmintics to control widespread drug-resistant gastrointestinal nematodes in ruminants, such as Haemonchus contortus. Artemisinin and its semi-synthetic derivatives are widely used against drug-resistant Plasmodium falciparum, but their role in veterinary medicine is only emerging. Artemisinin may be useful in controlling gastrointestinal parasites including Haemonchus. However, no ruminant studies involving artemisinin have been reported. The stability of artemisinin in capsules, crystals, or stock solutions in ethanol and dimethyl sulfoxide was evaluated in bovine rumen culture medium incubated for 24 hours at 39 degrees C. A second study established artemisinin kinetics in goats after oral administration of artemisinin capsules at 23 mg/kg of body weight. Artemisinin recovered from rumen culture ranged from 67 to 92% at pH 6.8 and was 95% at pH 3.0. The kinetics data showed that artemisinin was metabolized to dihydroartemisinin by goats, while unabsorbed artemisinin was eliminated in feces. Dihydroartemisinin peaked in the blood (0.7 mug/mL) at 12 hours, and decreased to 0.18 mug/mL at 24 hours. At 24 hours, artemisinin concentration in feces was 2.4 mug/g, indicating its poor bioavailability in goats when provided orally and as capsules. These results suggest that the bioavailability of artemisinin to goats can improve by dissolving capsules in ethanol or dimethyl sulfoxide, by using more stable and bioavailable artemisinin-derived drugs, and by using routes of delivery other than oral.