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Title: Anthelmintic activity of Artemisia annua L. extracts in vitro and the effect of an aqueous extract and artemisinin in sheep naturally infected with gastrointestinal nematodes

Author
item Cala, Aida - Mozambique Agronomic Research Institute
item Ferreira, Jorge
item Chagas, Ana Carolina - Embrapa
item Gonzalez, Javier
item Rodriguez, Rodney A. F. - Universidade De Campinas (UNICAMP)
item Foglio, Mary Ann - Universidade De Campinas (UNICAMP)
item Oliveira, Marcia C. S. - Embrapa
item Souza, Ilza M. O. - Universidade De Campinas (UNICAMP)
item Magalhaes, Pedro - Universidade De Campinas (UNICAMP)
item Barioni Junior, Waldomiro - Embrapa

Submitted to: Parasitology Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2014
Publication Date: 5/7/2014
Citation: Cala, A.C., Ferreira, J.F., Chagas, A.S., Gonzalez, J.M., Rodriguez, R., Foglio, M., Oliveira, M., Souza, I., Magalhaes, P.M., Barioni Junior, W. 2014. Anthelmintic activity of Artemisia annua L. extracts in vitro and the effect of an aqueous extract and artemisinin in sheep naturally infected with gastrointestinal nematodes. Parasitology Research. 113:2345-2353.

Interpretive Summary: Gastrointestinal nematodes (GIN) are the most important economic hindrance in small ruminants grazing systems worldwide, with the barber-pole worm being the most economically-important GIN. Currently, there is no effective natural alternative control for GIN, and resistance to most commercial anthelmintics has been reported worldwide. The objective of this study was to evaluate both in vitro and in vivo activities of crude extracts of Sweet Annie and in vivo activity of artemisinin (ART), the main anti-parasitic component of Sweet Annie, as natural anthelmintics. Different Sweet Annie extracts were evaluated in vitro, by the egg hatch test (EHT) and by the larval development test (LDT), against sheep GIN. Extracts were made with water, aqueous 0.1% sodium bicarbonate (ASBE), dichloromethane, and ethanol. Both ASBE (2g/kg BW) and ART (100 mg/kg BW) were evaluated in a single oral dose in Santa Inês sheep naturally infected with GIN. Parameters evaluated in vivo included packed cell volume (PCV) and nematode eggs excreted per gram (EPG) of feces. Although other nematodes were found in naturally infected sheep, 84-91% of the eggs belonged to the barber-pole worm. Analysis of lyophilized Sweet Annie water, ASBE, ethanol, and dichloromethane extracts determined that the extracts had 0.3%, 0.6%, 4.4%, and 9.8% of ART, respectively, while the ASBE used in vivo had 0.9% deoxyartemisinin, but no ART. Chemical analysis of ART capsules detected an average of 100 mg ART/capsule. ASBE also was rich in antioxidant activity. Both in vitro tests determined that the ASBE was the most lethal extract to barber-pole worm. In vivo, infected sheep treated with the ASBE or ART had EPG reductions of 19.0% and 50%, respectively, at day 15. While these EPG reductions were not significantly different from reduction provided by levamisole (94%), they were significantly smaller than that of infected, non-treated, sheep (106% increase in EPG). Non-absorbed ART in feces of sheep dosed with ART peaked at 24h after treatment. This is the first time the potential anthelmintic effect of oral ART is reported as a natural anthelmintic in sheep. These results suggest that both crude plant extracts rich in ART and ART analogs should be tested in vivo for their potential to control GIN in small ruminants.

Technical Abstract: There is no effective natural alternative control for gastrointestinal nematodes (GIN) of small ruminants, with Haemonchus contortus being the most economically important GIN. Despite frequent reports of multidrug-resistant GIN, there is no new commercial anthelmintic to substitute failing ones. Although trematocidal activity of artemisinin analogs has been reported in sheep, neither artemisinin nor its plant source (Artemisia annua) has been evaluated for anthelmintic activity in ruminants. This study evaluated the anthelmintic activity of A. annua crude extracts in vitro and compared the most effective extract with artemisinin in sheep naturally infected with H. contortus. A. annua leaves extracted with water, aqueous 0.1 % sodium bicarbonate, dichloromethane, and ethanol were evaluated in vitro by the egg hatch test (EHT) and with the bicarbonate extract only for the larval development test (LDT) using H. contortus. The A. annua water, sodium bicarbonate (SBE), ethanol, and dichloromethane extracts tested in vitro contained 0.3, 0.6, 4.4, and 9.8 % of artemisinin, respectively. The sodium bicarbonate extract resulted in the lowest LC99 in the EHT (1.27 µg/mL) and in a LC99 of 23.8 µg/mL in the LDT. Following in vitro results, the SBE (2 g/kg body weight (BW)) and artemisinin (100 mg/kg BW) were evaluated as a single oral dose in naturally infected Santa Inês sheep. Speciation from stool cultures established that 84–91 % of GIN were H. contortus, 8.4–15.6 % were Trichostrongylus sp., and 0.3–0.7 % were Oesophagostomum sp. Packed-cell volume and eggs per gram (EPG) of feces were used to test treatment efficacy. The SBE tested in vivo contained no artemisinin, but had a high antioxidant capacity of 2,295 µmol of Trolox equivalents/g. Sheep dosed with artemisinin had maximum feces concentrations 24 h after treatment (126.5 µg/g artemisinin), which sharply decreased at 36 h. By day 15, only levamisole-treated sheep had a significant decrease of 97 % in EPG. Artemisinin-treated and SBE-treated sheep had nonsignificant EPG reductions of 28 and 19 %, respectively, while sheep in infected/untreated group had an average EPG increase of 95 %. Sheep treated with artemisinin and A. annua SBE maintained blood hematocrits throughout the experiment, while untreated/infected controls had a significant reduction in hematocrit. This is the first time oral dose of artemisinin and an aqueous extract of A. annua are evaluated as anthelmintic in sheep. Although oral dose of artemisinin and SBE, at single doses, were ineffective natural anthelmintics, artemisinin analogs with better bioavailability than artemisinin should be tested in vivo, through different routes and in multiple doses. The maintenance of hematocrit provided by artemisinin and A. annua extract and the high antioxidant capacity of the latter suggest that they could be combined with commercial anthelmintics to improve the well-being of infected animals and to evaluate potential synergism.