In vitro anthelmintic evaluation of three alkaloids against gastrointestinal nematodes of goats.

This study assessed the in vitro anthelmintic activity of the alkaloids berberine, harmaline and piperine on gastrointestinal nematodes (GIN) of goat and their possible cytotoxic effects in Vero cells. The anthelmintic evaluation was performed using the egg hatch (EHA) and larval motility (LMA) assays. Cytotoxicity was determined using the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) assay. The alkaloids berberine and piperine inhibited the hatching of GIN eggs in more than 90 %. Piperine was the most active compound against goat GIN eggs with an EC50 (effective concentration 50 %) of 0.0074 mM (0.0021 mg/mL), while the EC50 of berberine was 1.32 mM (0.49 mg/mL). Harmaline (EC50 = 1.6 mM - 0.34 mg/mL) showed moderate ovicidal action (80.30 %). In LMA, piperine and harmaline reduced larval motility in 2.75 and 25.29 %, respectively. Larvicidal efficacy was evidenced only with the alkaloid berberine, which showed a percentage of inhibition of larval motility of 98.17 % (2.69 mM =1.0 mg/mL). In the MTT assay, all alkaloids showed low toxicity to Vero cells, with a percentage of cell viability greater than 50 % in all concentrations tested. These results suggest that berberine and piperine have anthelmintic potential on goat gastrointestinal nematodes with low toxicity to mammalian cells.

In vitro and in silico studies of the larvicidal and anticholinesterase activities of berberine and piperine alkaloids on Rhipicephalus microplus.

Rhipicephalus microplus is responsible for high economic losses in livestock and its control has become difficult due to the establishment of tick populations resistant to commercial acaricides. This study aimed to evaluate the in vitro larvicidal effect of the alkaloids berberine and piperine, and also to investigate their inhibitory mechanisms against the acetylcholinesterase enzyme. The effects of the alkaloids on larvae were observed through the immersion test at the following concentrations: 1.5; 3; 6; 12; 16 and 24 mM. Berberine and piperine presented larvicidal activity greater than 95 %, not differing from 100 % for the positive fipronil control (p > 0.05). Of the two alkaloids, piperine had a lower effective concentration (EC), with an EC50 of 6.04 mM. The acetylcholinesterase enzyme used in the study was obtained from R. microplus larvae (RmAChE) and the anticholinesterase activity was determined spectrophotometrically. The highest anticholinesterase activity, measured as inhibition concentration (IC), was observed for berberine (IC50 = 88.13 µM), while piperine showed lower activity (IC50 > 200 µM). Docking studies in RmAChE, followed by 10 ns molecular dynamics simulation, suggest that berberine stabilizes the RmAChE at lower Root-Mean-Square Deviation (RMSD) than Apo protein. Few hydrogen-bond interactions between berberine and RmAChE residues were balanced by hydrophobic and π-type interactions. Berberine fills preferentially the peripheral anionic site (PAS), which correlates with its non-competitive mechanism. These results suggest that berberine and piperine alkaloids have an in vitro acaricidal action on R. microplus larvae, and the likely mechanism of action of berberine is related to RmAChE inhibition when accessing the PAS residues. These findings could help the study of new natural products that could inhibit RmAChE and aid in the development of new acaricides.

In vitro anthelmintic and cytotoxicity activities the Digitaria insularis (Poaceae).

This study aimed to evaluate the in vitro activity of D. insularis extracts and fractions against gastrointestinal nematodes of goats and its cytotoxicity on Vero cells. The egg hatch (EHT) and larval motility (LMT) tests were conducted to investigate the anthelmintic effects of the crude hydroethanolic (CH), ethyl acetate (EA), butanolic (BT) and residual hydroethanolic (RH) extracts. The elution of the active extract (EA) on column chromatography (SiO2) using organic solvents furnished six fractions (FR1 to FR6), which were also tested. Cytotoxicity was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan Blue exclusion assays. All extracts, FR2 and FR3, inhibited egg hatching in a concentration-dependent manner. The EHT led to EC50 values (effective concentration 50%) of 0.64; 0.69; 0.77; 0.96; 0.27 and 0.65mg/mL for CH, EA, BT, RH, FR2 and FR3, respectively. However, the extracts exhibited low effect on the motility of L3. In the cytotoxicity evaluation (MTT assay), the IC50 (inhibitory concentration 50%) was 1.18 (EA), 1.65 (FR2) and 1.59mg/mL (FR3), which was relatively high (low toxicity) in comparison to the EC50 values in EHT, mainly for FR2. The chemical analyses of most active fractions (FR2) by Liquid Chromatography coupled to Mass Spectrometry (LC-MS) led the characterization of the flavones tricin and diosmetin. These results showed the high anthelmintic effect and low cytotoxicity of D. insularis and also that the flavones can be probably responsible for the nematocidal activity of this plant.

In vitro anthelmintic activity of the Zizyphus joazeiro bark against gastrointestinal nematodes of goats and its cytotoxicity on Vero cells.

This study examined the in vitro effect of the Zizyphus joazeiro bark against gastrointestinal nematodes of goats and its cytotoxicity on Vero cells. The ovicidal activity of the crude hydroethanolic extract (CE), its partitioned hexane (HE) and aqueous extract (AE) and saponins fraction (SF), including betulinic acid (BA), a biogenetic compound from this plant found in HE, were investigated using the inhibition of egg hatch assay (EHA). Thereafter, the extracts and the SF were evaluated through the larval motility assay (LMA) and larval migration inhibition assay (LMIA). The AE and SF promoted a complete inhibition of the egg hatch, and the effective concentration to inhibit 50% (EC50) values was 1.9 and 1.3mg/mL, respectively. The highest percentages of inhibition in EHA observed after treatments with CE, HE and BA corresponded to 79, 48 and 17%, respectively. The extracts and SF did not show larvicidal activity in LMA and LMIA. The AE and SF demonstrated cytotoxic effects in 3-4,5-dimethylthiazol-2-yl, 2,5diphenyltetrazolium bromide (MTT) and trypan blue tests; however, SF was more toxic (50% inhibitory concentration, IC50=0.20mg/mL). The chemical characterization of the SF was made through Proton Nuclear Magnetic Resonance ((1)H NMR) and Electrospray Ionization Mass Spectrometry (ESI-MS) analyses, which led to the identification of two saponins known as Joazeiroside B and Lotoside A. The results obtained from the research of this saponin content provide important information about the biological activity, especially the anthelmintic effect present in the plant investigated. That also suggests the types of bioactive compounds that may be responsible for this antiparasitic activity exhibited by the plant extracts.