IN VITRO EFFECTS OF HYDRO-METHANOLIC EXTRACT FROM Gliricidia sepium LEAVES ON LARVAE OF Haemonchus sontortus

The aim of this in vitro study was to evaluate the anthelmintic effects of extracts of Gliricidia sepium on sheathed and exsheathed larvae of Haemonchus contortus. Larvae of this parasite were incubated at 20–25 °C in hydro-methanolic extracts of leaves from this tropical tree at concentrations of 12.5, 25, 50, 100, and 200 mg/mL for 24, 48, or 72 h. Water and ivermectin were negative and positive controls, respectively. Total phenolic compounds of leaves of G. sepium were 6.4 ± 2.4 mg/g of dry matter. Other compounds identified in this leguminous tree by HPLC-mass spectrometry and that may be responsible for the anthelmintic effects observed were vanillin 4-sulfate, prodelphinidin p-coumaroyl glucose, kaempferol 3-o-glucosyl-rhamnosyl-glucoside,kaempferol-3-O-xylosyl rutinoside, p-coumaric acid, luteolin 7-rutinoside, isorhamnetin 3-glucoside-7-rhamnoside, and dihydro ferulic acid. At doses of 100 mg/mL mortality rate of sheathed and exsheathed H. contortus was 21.6 and 44.7%, respectively for 72 h of incubation. At 200 mg/mL, the hydro-methanolic extracts of G. sepium killed 61.5 and 93.8% of sheathed and exsheathed larvae, respectively, after 72 h of incubation. The effective concentration of the G. sepium extract for 50% sheathed and exsheathed larvae mortality (EC50) after 72 h of incubation was 74 mg/mL (CI = 46–100) and 68 mg/mL (CI= 32–100), respectively. The significant (P<0.001) ability to kill larvae compared to the negative controls, suggests in vitro anthelmintic properties of G. sepium against H. contortus. UČINKI HIDROMETANOLNEGA IZVLEČKA LISTOV Gliricidia sepium NA LIČINKE Haemonchus contortus IN VITRO  Izvleček: Namen te in vitro študije je bil oceniti protiglivične učinke izvlečkov Gliricidia sepium na ličinke Haemonchus contortus z ovojem in brez njega. Ličinke parazita so bile inkubirane 24, 48 ali 72 ur pri 20–25 °C v hidrometanolnih izvlečkih listov tega tropskega drevesa v koncentracijah 12,5, 25, 50, 100 in 200 mg/ml. Voda in ivermectin sta služila kot negativna in pozitivna kontrola. Skupne fenolne spojine v listih G. sepium so obsegale 6,4 ± 2,4 mg/g suhe snovi. Druge spojine, ki so bile v drevesu identificirane s HPLC-masno spektrometrijo in ki bi lahko bile odgovorne za opažene protiglivične učinke, so bile vanilin 4-sulfat, prodelfinidin p-kumaroil glukoza, kaempferol 3-O-glukozil-rimnozil-glukozid, kaempferol-3-O-ksilozil rutinozid, p-kumarna kislina, luteolin 7-rutinozid, izorhamnetin 3-glukozid-7-rimnozid in dihidro ferulinska kislina. Pri odmerkih 100 mg/ml in 72 urah inkubacije je bila stopnja smrtnosti pri H. contortus z ovojem 21,6 %, pri H. contortus brez ovoja pa 44,7 %. Pri odmerkih 200 mg/ml in 72 urah inkubacije so hidrometanolni izvlečki G. sepium uničili 61,5 % ličink z ovojem in 93,8 % ličink brez ovoja. Srednja efektivna koncentracija (EC50) izvlečka G. sepium za ličinke z ovojem je bila 74 mg/ml (CI  =  46–100), za ličinke brez ovoja pa 68 mg/ml (CI  =  32–100) po 72 urah inkubacije. Statistično značilna (P < 0,001) sposobnost uničenja ličink v primerjavi z negativno kontrolo kaže na protiglivične lastnosti G. sepium proti H. contortus in vitro. Ključne besede: flavonoidi; flavonoli; ličinke; nematodi; tanini