Polyphenolic extract from Punica granatum peel causes cytoskeleton-related damage on Giardia lamblia trophozoites in vitro

Plant material and processing

Pomegranates (Punica granatum L.) were commercially acquired in Cuatrociénegas, Coahuila, México. The collected vegetable sample was cleaned and dehydrated in an oven at 50 °C, later the material was grinded until a reduction of the particle size of 0.6 mm.

Phytochemical extraction using microwave/ultrasound

The ethanolic P. granatum extract was obtained using a mass/volume (m/v) relation of 1:12. In this step, 83.33 g of the dry sample was resuspended in 1 L of 30% ethanol-water. The solution was transferred to a 1.5 L crystal reactor and placed in an Ultrasonic Microwave Reaction System (Nanjing ATPIO Instruments Manufacture Co. Ltd., Nanjing, China) under the conditions described before by Valdez-Guerrero and cols (Yathzamiry et al., 2021): Power Radio 20, Ultrasonic on Relay 10, Ultrasonic Off Relay 3, Amplitude Transformer 25, and Set 20 for Ultrasound and Power Radio 800, Display Power 0, Set Time 70 °C and Holding Time 5 for Microwaves. After this treatment, the ethanolic extract was filtered using Whatman No.41 filter paper (Cytiva, Uppsala, Sweden) and stored at 4 °C in amber recipients.

Polyphenolic fraction obtained by Amberlite

The polyphenolic fraction was obtained in a chromatographic column (Merck KGaA, Darmstadt, Germany) where Amberlite XAD-16 resin (MilliporeSigma, Darmstadt, Germany) was used as a stationary phase. The ethanolic P. granatum extract was passed through the column, using distilled water for the removal of carbohydrates, lipids, and other impurities. Later, the phenolic fraction was recuperated from the resin using 96% ethanol as eluent. After that, the ethanol was evaporated from the phenolic fraction using an oven at 45 °C for 12 h until a fine powder was obtained (Hernández-Hernández et al., 2020). The polyphenolic powder was stored in amber bottles at 4 °C to avoid compound degradation.

Identification of polyphenolic compounds by HPLC/MS

The characterization of PP polyphenolic fractions was performed by reversed-phase high-performance liquid chromatography using an HPLC system Varian ProStar (Agilent, Santa Clara, CA, USA) with a diode array detector (280 nm) and mass spectrometer (MS) with a liquid chromatography ion trap Varian 500-MS IT (Agilent, Santa Clara, CA, USA) equipped with an electrospray ion source. The sample (2 mg) was dissolved in 1 mL of 96% ethanol, filtered by 0.45 µm nylon membranes and injected into a Denali C18 column (150 × 2.1 mm, 3 µm, Grace, Albany, OR, USA) maintaining a temperature at 30 °C. The eluents were formic acid (0.2%, v/v) and acetonitrile. The conditions of elution steps were made according to (Ascacio-Valdés et al., 2016). The MS experiments has been made in the negative mode [M-H]-using nitrogen and helium as nebulizing gas and damping gas, respectively. The ion source parameters were spray voltage 5.0 kV and capillary voltage, and temperature were 90.0 V and 350 °C, respectively. The collection and process of the data were done using MS Workstation software Version 6.9 (Agilent, Santa Clara, CA, USA). Sample was analyzed in full scan mode acquired in the m/z range 50–2,000. MS/MS analyses were performed on a series of selected precursor ions. Compounds identification was performed comparing the results with a database of bioactive compounds (WorkStation version 2.0 database, VARIAN, Palo Alto, CA, USA) (Ascacio-Valdés et al., 2016).

Giardia culture and growth inhibition assay

Trophozoites of G. lamblia (ATCC 50803) were axenically grown at 37 °C in borosilicate culture tubes (Thermo Fisher Scientific, Waltham, MA, USA) containing TYI-S-33 medium, pH 7.1, supplemented with 10% bovine serum and 0.5 mg/mL bovine bile (Keister, 1983). Cultures were maintained by subculturing twice a week. In order to determine the effect of polyphenolic extract from P. granatum on G. lamblia trophozoites growth, an inoculum of 350,000 cells from log-phase of growth were incubated for 24, 48 and 72 h at 37 °C with different concentrations of the polyphenolic extract (0, 125, 150, 175, and 200 µg/mL), 0.1% dimethyl sulfoxide (DMSO) (MilliporeSigma, Darmstadt, Germany) and 1.5 µg/mL metronidazole (MTZ) (PiSA, Guadalajara, Mexico) were used as diluent and positive control, respectively. After the incubation times, trophozoites were detached by 30 min of cooling in an ice-water bath and counted by optical microscopy (Carl Zeiss AG, Oberkochen, Germany) using a hemocytometer. Experiments were performed by triplicate and repeated at least twice. Results were expressed as the total number of cells and as the percentage of trophozoites growth in comparison with the control tube (without treatment), also, the 50% inhibitory concentration (IC₅₀) values were calculated by regression analysis.

Adherence inhibition assay

To determine the effect of polyphenolic extract on the capacity of G. lamblia trophozoites to attach into inert superficies, cells treated with P. granatum extract at 0, 125, 150, 175, and 200 µg/mL, and 0.1% dimethyl sulfoxide (DMSO) (MilliporeSigma, Darmstadt, Germany) were grown at 24, 48 and 72 h. After these times, medium with non-adhered trophozoites was removed and maintained on ice for 30 min, after this, tubes were filled with phosphate buffered saline (PBS) and cooled on ice bath for 30 min to detach the adhered cells. The number of trophozoites (adhered and non-adhered) were counted by optical microscopy using a hemocytometer. Results were expressed as percentage of adherence inhibition in relation to the control tube. Experiments were performed by triplicate and repeated at least twice.

The effect of pomegranate peel extract on morphology by Scanning Electron Microscopy (SEM)

Morphologic damage alterations were analyzed using trophozoites incubated for 48 h in 0.1% dimethyl sulfoxide (DMSO) (MilliporeSigma, Darmstadt, Germany) and treated with 150, 175, and 200 µg/mL of the PP extract. Cells were collected, PBS-washed, fixed with 2.5% glutaraldehyde (MilliporeSigma, Darmstadt, Germany) in PBS for 1 h, and adhered to 0.2% poly-(ethylenimine) (MilliporeSigma, Darmstadt, Germany)-coated coverslips. Fixed trophozoites were dehydrated using ethanol increasing concentrations (50–100% v/v) and dried to critical point with CO_2. Samples were coated with a thin layer of gold and analyzed by FE-SEM-Hitachi (Hitachi High-Technologies Corporation, Tokyo, Japan).

Effect of pomegranate peel extract on α-tubulin expression

Trophozoites 48 h-treated with 150, 175, and 200 µg/mL of PP extract or 0.1% dimethyl sulfoxide (DMSO) (MilliporeSigma, Darmstadt, Germany) were used to obtain total protein extracts using the NP-40 buffer (MilliporeSigma, Darmstadt, Germany). The protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and 10 µg of the protein samples were separated by 10% (w/v) SDS-PAGE using Tris-glycine buffer (Santa Cruz Biotechnology, Dallas, TX, USA) and transferred to PVDF membranes. Protein transfer was performed in PVDF membrane (Millipore, Burlington, VT, USA) using the Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad Laboratories, Hercules, CA, USA). Membrane was blocked with 5% BSA/0.1% PBS-Tween (PBST) (FAGA-Lab, Sinaloa, Mexico) solution for 1.5 h at room temperature, washed with PBST 0.1% and incubated with primary antibody (1/500 mouse anti-α-tubulin) (Santa Cruz Biotechnology, Dallas, TX, USA) for 18 h. Membrane was washed again with PBST and incubated for 1 h with 1/5,000 of anti-mouse m-IgGκ BP-HRP (horseradish peroxidase) secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Membrane was washed with PBST and the signal was detected by chemiluminescence using the kit Immobilon ECL Ultra Western HRP Substrate (Millipore, Burlington, VT, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used as protein loading control (1/200) (Yang et al., 2002). Experiments were done by triplicate.

The images were captured with the Vision Works version 8.17.16133.9147 Software by Analytik Jena 2016 (www.analytik-jena.de). Semi-quantitative evaluation was performed by densitometry, the GelQuant.NET version 1.8.2 software (BiochemLabSolutions.com, San Francisco, CA, USA) was used.

Effect of pomegranate peel extract on α-tubulin distribution

To analyze the pattern of α-tubulin distribution, trophozoites treated 48 h with 125, 150, 175, and 200 µg/mL of PP extract, DMSO, or albendazole 0.5 µM (MilliporeSigma, Darmstadt, Germany) were analyzed by immunofluorescence (IFA). Samples were collected and counted in a hemocytometer to allow 500,000 cells to adhere in coverslips treated with 0.2% poly-(ethylenimine) (MilliporeSigma, Darmstadt, Germany) for 30 min at 37 °C. Trophozoites were fixed using methanol for 10 min at −20 °C (MilliporeSigma, Darmstadt, Germany). Once dried, the samples were permeabilized in 0.5 Triton X-100 (MilliporeSigma, Darmstadt, Germany) 15 min, and blocked with 1% bovine serum albumin (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. Later, samples were incubated 2 h at room temperature with 1/200 mouse anti-α-tubulin IgG₁ƙ monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), washed three times with PBS, and incubated 1 h with 1/200 anti-mouse IgGκ light chain binding protein (m-IgGκ BP) conjugated to fluorescein isothiocyanate (FITC) (Santa Cruz Biotechnology, Dallas, TX, USA), and washed twelve times with PBS. Mounting of the preparations were done using Prolong Gold with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Preparations were analyzed by fluorescence Axiolab A1 35 LED Binocular Microscope (Carl Zeiss™, Oberkochen, Germany) and image acquisition was done using the software, ZEN 2.3 edition.

Statistical analysis

The data from growth, adherence inhibition and western blot were analyzed by GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) using the two-way ANOVA test and as post hoc the Tukey test, statistical significance was considered with P values ≤ 0.05.

Identification of polyphenolic compounds in pomegranate peel extract by HPLC/MS

Phytochemical isolation was performed by microwave-ultrasound hybridization using ethanol as a solvent. The obtention of enriched polyphenolic extract was realized by liquid chromatography with Amberlite XAD-16. The chromatograms obtained through HPLC system and mass spectrometer were compared on a database of bioactive compounds. The characterization of pomegranate peel showed the presence of ellagitannins (punicalin and punicalagin isomers, galloyl-dihexahydroxydiphenoyl-hexoside), flavones (luteolin 6-C-glucoside), and ellagic acid (including ellagic acid-hexoside) (Table 1).

Polyphenolic extract effects on Giardia lamblia trophozoite growth

Results showed that the tested extract inhibited the parasite growth, all the extract concentrations used in this study were statistically different in comparison to non-treated cells at the times tested. The effect was evident at 24 h, but at 48 h, it was observed the maximal effect in comparison with negative control. There was no difference between non-treated cells and cells grown with the diluent of the extract. Trophozoites treated with 200 µg/mL of pomegranate peel extract showed an effect like MTZ at 48 and 72 h (69.09% and 68.15% of inhibition, respectively), showing significant growth inhibition of 74.36% at 48 h and 66.32 at 72 h (Fig. 1). The calculated IC₅₀ of the extract was 179 µg/mL at 48 h. These results demonstrate that the polyphenolic extract from the studied vegetable source, has an in vitro inhibitory effect on G. lamblia growth.

Polyphenolic extract affects trophozoite adherence to inert surfaces

To establish if the studied extract influences trophozoite adherence to inert surfaces, growth kinetics were performed, treating trophozoites with the polyphenolic extract. Results showed that the extract reduced adherence since 24 h, except for 125 µg/mL, in comparison with cells without treatment. The higher effect on adherence inhibition was observed at 48 h in a dose-dependent manner, where the 200 µg/mL treatment was able to inhibit G. lamblia trophozoite adherence in 46.8% (Fig. 2). These results demonstrate that the tested polyphenolic extract was able to in vitro inhibit G. lamblia trophozoite adherence.

Pomegranate peel extract alters the morphology on Giardia lamblia trophozoites

To evaluate alterations in G. lamblia trophozoite morphology caused by the extract; cells were treated with polyphenolic pomegranate peel extract and DMSO as negative control for 48 h, then, they were visualized using SEM. Parasites grown in solvent showed normal pear shape (Fig. 3A). In treated cells, an elongation, loss of shape, and protrusions on the dorsal surface were observed. Trophozoites incubated with the extract had irregularities on the periphery and showed vesicle-like structures on the dorsal surface. In addition, abnormalities on the caudal region and flagella deformations, including shortening or loss, compared with control cells were detected (Figs. 3B–3D). Pomegranate extract also appears to cause holes on the membrane (perforation), this effect increased with concentration (Fig. 3D).

Tubulin G. lamblia alteration by pomegranate peel extract

In order to determine changes in cytoskeleton protein expression, α-tubulin was detected by Western Blot. It was found an alteration on α-tubulin expression levels by pomegranate extract treatment in trophozoites with 150 and 200 µg/mL in comparison with DMSO incubated cells. Interestingly, it is observed that treatments affect α-tubulin expression in a concentration-independent manner (Fig. 4), where the lowest concentration decreased protein expression, but the highest concentration increased it. At 175 µg/mL no differences were found. Another additional finding is shown in Fig. S1 where glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was attempted to be used as a loading control, however, the samples treated with the PP extract increased the expression of this protein.

Tubulin distribution of G. lamblia was affected by pomegranate peel extract

Immunofluorescence assay was performed in order to analyze alterations on the cell localization of α-tubulin on PP treated trophozoites. In non-treated cells (Fig. 5A–A2), α-tubulin is located on flagella, median body and ventral disc (VD), in addition, cells present normal size and shape. Conversely, cells treated with the extract showed differences in the staining pattern, which indicates modifications in G. lamblia microtubule cytoskeleton. Interestingly, these alterations are independent of the concentration, like WB observations. Immuno-labeling of cells treated with PP extract showed interesting events on VD distribution. In 125 μg/mL treated cells with the ventral side exposed, the α-tubulin signal in VD is not localized, nor the bare area is observed (Fig. 5–B2). At 175 µg/mL and 200 µg/mL the intensity of tubulin is lower than control, VD appears incomplete (Fig. 5–D2, asterisk), with diffuse outline, and incomplete cells consisting only of the disc (Fig. 5–E2), were observed. These changes are associated with morphological alterations, where a loss of characteristic pear shape and VD deformation was observed (Fig. 5B–E). In accordance with SEM, flagellar disturbance as bifurcation (Fig. 5–C2, arrow) or structure loss (Fig. 5–E2) were also detected. For ABZ exposure, trophozoites appear completely deformated and increased in size (Fig. 5F-F2).