Acaricidal activity of Acmella oleracea (Asteraceae) extract against Rhipicephalus microplus: What is the influence of spilanthol?

The present study was carried out to evaluate and compare the acaricidal activity of different fractions of Acmella oleracea methanolic extract, containing 0.0 % (F1), 24.5 % (F2), 48.0 % (F3) and 100 % (F4) of spilanthol, on unfed larvae and engorged females from the same Rhipicephalus microplus population. To obtain these fractions, the crude extract was subjected to different extraction procedures using increasingly polarized solvents to isolate the spilanthol compound. The Larval Packet Test was used to evaluate acaricidal activity in unfed larvae at concentrations ranging from 0.2 to 25.0 mg/mL, while for engorged females, the Adult Immersion Test was performed at concentrations from 3.1 to 25.0 mg/mL. The F1 fraction showed no activity on unfed larvae, while a control percentage of 44.6 % was observed at a concentration of 25.0 mg/mL for engorged females. For unfed larvae, the F2 fraction resulted in 95.7 % mortality at a concentration of 1.6 mg/mL, with a control percentage of 92.7 % for engorged females at a concentration of 12.5 mg/mL. Fractions F3 and F4 had similar activity against unfed larvae, with mortality >84.0 % from the concentration of 0.8 mg/mL. This similarity between the fractions was also observed for engorged females from a concentration of 12.5 mg/mL, resulting a control percentage >94.0 %. These results demonstrate that the presence of spilanthol is an important factor for the acaricidal activity of A. oleracea extract. Fraction extracts with 24.5, 48 and 100 % of spilanthol have similar acaricidal activity on R. microplus.

Activity of the extract of Acmella oleracea on immature stages of Amblyomma sculptum (Acari: Ixodidae).

This study evaluated the acaricidal activity of the methanol extract of Acmella oleracea with 0.187% of spilanthol against immature stages of Amblyomma sculptum. The packet test was used to evaluate the extract's activity on unengorged larvae and nymphs, testing concentrations of 0.4 to 50 mg/mL for larvae and 12.5 to 200.0 mg/mL for nymphs. For the engorged stages, the immersion test was used, at concentrations of 0.4 to 50 mg/mL for larvae and 12.5 to 200.0 mg/mL for nymphs. The methanol extract caused 100% mortality of the unengorged larvae and nymphs starting at concentrations of 12.5 and 200.0 mg/mL, respectively. For engorged larvae and nymphs, the mortality was 100% starting from concentrations of 12.5 and 150.0 mg/mL, respectively. The LC50 for unengorged larvae was 3.2 mg/mL, while for engorged larvae it was 6.6 mg/mL. For unengorged nymphs, the LC50 was 38.5 mg/mL, but it was not possible to calculate the corresponding value for engorged nymphs because the data did not fit the probit model. These results demonstrate that the methanol extract of A. oleracea has acaricidal activity against different immature stages of A. sculptum.

Acaricidal activity of methanol extract of Acmella oleracea L. (Asteraceae) and spilanthol on Rhipicephalus microplus (Acari: Ixodidae) and Dermacentor nitens (Acari: Ixodidae).

We evaluated the acaricidal activity of Acmella oleracea methanol extract and spilanthol on Rhipicephalus microplus and Dermacentor nitens. The extract was made through maceration with methanol. From this extract, a dichloromethane fraction with 99% spilanthol was obtained and tested on R. microplus larvae and engorged females and D. nitens larvae. For evaluation against larvae, the modified larval packet test was used, and both the methanol extract and dichloromethane fraction were tested at concentrations of 0.2-50mg/mL. The modified larval packet test was also used in the lethal time (LT) test, with the methanol extract at a concentration of 12.5mg/mL and the percentage mortality was assessed after 15, 30, 45, 60, 75, 90, 120min and 24h. The 50% lethal time calculation (LT50) was performed in this test. The engorged female test was performed with R. microplus only, at concentrations of 25-200mg/mL for methanol extract and 2.5-20.0mg/mL for spilanthol. The methanol extract caused 100% mortality of the R. microplus and D. nitens larvae at concentrations of 3.1 and 12.5mg/mL, respectively. Spilanthol resulted in 100% mortality of R. microplus larvae at concentration of 1.6mg/mL and of D. nitens at 12.5mg/mL. In the lethal time assay using the methanol extract, the mortality rate was 100% for R. microplus and D. nitens larvae after 120min and 24h, with LT50 values of 38 and 57min, respectively. In the test of females, the egg mass weight and the hatching percentage of the groups treated with concentrations equal to or higher than 50.0mg/mL of methanol extract were significantly reduced (p<0.05), while for spilanthol, the reduction of the egg mass weight and hatching percentage occurred from concentrations of 10.0mg/mL and 2.5mg/mL, respectively. Females treated with 200.0mg/mL of extract died before starting oviposition, resulting in 100% effectiveness, while the best efficacy for spilanthol was 92.9% at a concentration of 20.0mg/mL. Thus we conclude that the methanol extract of A. oleracea and spilanthol have acaricidal activity against R. microplus and D. nitens.

Spilanthol from Acmella Oleracea Lowers the Intracellular Levels of cAMP Impairing NKCC2 Phosphorylation and Water Channel AQP2 Membrane Expression in Mouse Kidney.

Acmella oleracea is well recognized in Brazilian traditional medicine as diuretic, although few scientific data have been published to support this effect. Aim of this study was to determine the molecular effect of Acmella oleracea extract and its main alkylamide spilanthol on two major processes involved in the urine concentrating mechanism: Na-K-2Cl symporter (NKCC2) activity in the thick ascending limb and water channel aquaporin 2 accumulation at the apical plasma membrane of collecting duct cells. Phosphorylation of NKCC2 was evaluated as index of its activation by Western blotting. Rate of aquaporin 2 apical expression was analyzed by confocal laser microscopy. Spilanthol-induced intracellular signalling events were dissected by video-imaging experiments. Exposure to spilanthol reduced the basal phosphorylation level of NKCC2 both in freshly isolated mouse kidney slices and in NKCC2-expresing HEK293 cells. In addition, exposure to spilanthol strongly reduced both desmopressin and low Cl--dependent increase in NKCC2 phosphorylation in mouse kidney slices and NKCC2-expressing HEK293 cells, respectively. Similarly, spilanthol reduced both desmopressin- and forskolin-stimulated aquaporin 2 accumulation at the apical plasma membrane of collecting duct in mouse kidney slice and MCD4 cells, respectively. Of note, when orally administered, spilanthol induced a significant increase in both urine output and salt urinary excretion associated with a markedly reduced urine osmolality compared with control mice. Finally, at cellular level, spilanthol rapidly reduced or reversed basal and agonist-increased cAMP levels through a mechanism involving increases in intracellular [Ca2+]. In conclusion, spilanthol-induced inhibition of cAMP production negatively modulates urine-concentrating mechanisms thus holding great promise for its use as diuretic.