Polyphenolic extract from Punica granatum peel causes cytoskeleton-related damage on Giardia lamblia trophozoites in vitro
Authors: Palomo-Ligas Lissethe , Estrada-Camacho Job , Garza-Ontiveros Mariana , Vargas-Villanueva José Roberto , Gutiérrez-Gutiérrez Filiberto , Nery-Flores Sendar Daniel , Cañas Montoya Jorge Arturo , Ascacio-Valdés Juan , Campos-Muzquiz Lizeth Guadalupe , Rodriguez-Herrera Raul 
Published: April 2022

Background
Diarrheal diseases caused by protozoa have a great impact on human health around the world. Giardia lamblia is one of the most common flagellates in the intestinal tract. Factors such as adverse effects to first-line drugs or the appearance of drug-resistant strains, make it necessary to identify new treatment alternatives. Agroindustry waste, like pomegranate peel, are a source of phenolic compounds, which possess antiparasitic activities. In vivo studies demonstrated antigiardiasic potential by reducing cyst shedding and protecting intestinal cells; however, they did not identify the compounds or elucidate any mechanism of action in the parasite. The objective of this study is to identify potential molecular targets and to test the in vitro effects of polyphenols from Punica granatum on Giardia lamblia.

Methods
The in vitro antigiardial potential of polyphenolic extract from pomegranate peel (Punica granatum L.) obtained using microwave-ultrasound methodology was evaluated on Giardia lamblia trophozoites. Extract phytochemical identification was performed by HPLC/MS analysis. The effect of polyphenolic extract on growth and adhesion capacity was determined by parasite kinetics; morphological damage was evaluated by SEM, alteration on α-tubulin expression and distribution were analyzed by western blot and immunofluorescence, respectively.

Results
The pomegranate peel extract showed the presence of ellagitannins (punicalin and punicalagin, galloyl-dihexahydroxydiphenoyl-hexoside), flavones (luteolin), and ellagic acid, that caused an inhibitory effect on growth and adhesion capacity, particularly on cells treated with 200 µg/mL, where growth inhibition of 74.36%, trophozoite adherence inhibition of 46.8% and IC50 of 179 µg/mL at 48 h were demonstrated. The most important findings were that the extract alters α-tubulin expression and distribution in Giardia trophozoites in a concentration-independent manner. Also, an increase in α-tubulin expression at 200 µg/mL was observed in western blot and diffuse or incomplete immunolabeling pattern, especially in ventral disk. In addition, the extract caused elongation, disturbance of normal shape, irregularities in the membrane, and flagella abnormalities.

Discussion
The pomegranate peel extract affects Giardia trophozoites in vitro. The damage is related to the cytoskeleton, due to expression and distribution alterations in α-tubulin, particularly in the ventral disk, a primordial structure for adhesion and pathogenesis. Microtubule impairment could explain morphological changes, and inhibition of adhesion capacity and growth. Besides, this is the first report that suggests that ellagic acid, punicalin, punicalagin and luteolin could be interactioning with the rich-tubulin cytoskeleton of Giardia. Further investigations are needed in order to elucidate the mechanisms of action of the isolated compounds and propose a potential drug alternative for the giardiasis treatment.