In Vitro Anthelminthic Efficacy of Aqueous Pomegranate (Punica granatum L.) Extracts against Gastrointestinal Nematodes of Sheep

Abstract

The worldwide increased difficulty to counteract gastrointestinal nematode (GIN) infection in sheep, due to progressing anthelmintic resistance, has led to the evaluation of other alternative helminth control options, mainly from plants. The anthelmintic efficacy of an aqueous Punica granatum macerate was evaluated in sheep naturally infected by GIN in southern Italy. The macerate was chemically characterized by chromatographic analysis coupled with high-resolution mass spectrometry (LC/HRMS) and an aliquot was concentrated to obtain a dry extract. A part was characterized, the remaining washed with methanol to obtain an insoluble residue and methanol phase. In the methanol fraction, the quantitatively predominant gallic acid was purified to obtain the pure molecule. The three fractions thus obtained were used for in vitro studies (i.e., egg hatch test) to verify anthelmintic efficacy. For this purpose, fecal samples were collected from sheep naturally infected by GINs. Fractions were diluted in H2O/DMSO 0.5% at 1.00, 0.5, 0.25, 0.125, 0.05, and 0.005 mg/mL concentrations. Thiabendazole (0.25 and 0.5 mg/mL) and deionized water were used as positive and negative controls, respectively. Egg hatch test results indicated that all fractions caused a significant (p < 0.05) egg hatch inhibition within 48 h of exposure highlighting a high (>82%) efficacy in vitro at all tested doses. Maximal egg hatching inhibition effect was exhibited by the methanol fraction (99.3% and 89.3% at 1 and 0.005 mg/mL concentrations), followed by the insoluble residue and gallic acid (94.7% and 85.3% and 94.0% and 82.7% at 1 and 0.005 mg/mL, respectively). The current study validated the anthelmintic potential of traditional P. granatum macerate against GIN infection in sheep, thus highlighting the role of gallic acid as principal component and justifying a need to undertake further in vivo studies on these ethno-veterinary remedies.

Green Veterinary Pharmacology Applied to Parasite Control: Evaluation of Punica granatum, Artemisia campestris, Salix caprea Aqueous Macerates against Gastrointestinal Nematodes of Sheep

“…On the contrary, a very low FECR% was observed in the other groups, namely TG2 ( A. campestris) and TG3 ( S. caprea) . The most effective P. granatum macerate, which was previously in vitro tested against GINs egg hatch test (EHT) [ 25 ], was as well chemically characterized for its composition as resumed in Table 3 presented in Section 4 .…”

“…The macerates were not concentrated, but just filtered and the average yield was of around 70% of the entire starting amount. The only effective macerate ( Punica granatum ) was analyzed using liquid chromatography electrospray ionization mass spectrometry (LC/MS-ESI) as previously described [ 25 ].…”

“…Also, its juice contains active compounds like polyphenols, tannins, anthocyanins, including vitamin C, tocopherols, lipoic acid, and punicalagin, bioactive constituents responsible for more than 50% of the antioxidant activity of pomegranate juice [ 24 ]. The anti-parasitic properties of this plant have been previously documented both in vitro [ 25 , 26 ] and in vivo [ 27 ] in animal models, and in its extract was documented the presence of compounds gallic acid and ellagic acid, which might explain its anthelmintic efficacy. However, its in vivo efficacy in small ruminants, still needs to be tested.…”

Antinematode Activity of Abomasum Bacterial Culture Filtrates against Haemonchus contortus in Small Ruminants

“…It is not advisable to eliminate the parasites from livestock, but to keep the population under a threshold, in a sustainable state [ 16 ]. To control the nematode parasites, different management practices, such as the use of chemical anthelmintics, sanitation, vaccination, various plant extracts, and biological control, are in common practice [ 17 ].…”

Natural Products Are a Promising Source for Anthelmintic Drug Discovery

“…A growing area of investigation is the identification of new anthelmintics from plant extracts. Several extracts from plants have shown potential for development as anthelmintic agents [85][86][87][88][89][90] and the screening attempts and scientific validation of the number of products with anthelmintic potential has continued over the past twenty years (Figure 3). This increased attention on plant extracts for the identification of new anthelmintics is likely to significantly increase in the future as anthelmintic resistance to current drugs increases.…”