In Vitro Studies Reveal Antiurolithic Effect of Antioxidant Sulfated Polysaccharides from the Green Seaweed Caulerpa cupressoides var flabellata

Abstract

Urolithiasis affects approximately 10% of the world population and is strongly associated with calcium oxalate (CaOx) crystals. Currently, there is no efficient compound that can be used to prevent this disease. However, seaweeds' sulfated polysaccharides (SPs) can change the CaOx crystals surface's charge and thus modify the crystallization dynamics, due to the interaction of the negative charges of these polymers with the crystal surface during their synthesis. We observed that the SPs of Caulerpa cupressoides modified the morphology, size and surface charge of CaOx crystals. Thus, these crystals became similar to those found in healthy persons. In the presence of SPs, dihydrate CaOx crystals showed rounded or dumbbell morphology. Infrared analysis, fluorescence microscopy, flow cytometry (FITC-conjugated SPs) and atomic composition analysis (EDS) allowed us to propose the mode of action between the Caulerpa's SPs and the CaOx crystals. This study is the first step in understanding the interactions between SPs, which are promising molecules for the treatment of urolithiasis, and CaOx crystals, which are the main cause of kidney stones.

Keywords: COD dumbbell; COD stabilization; green algae; urolithiasis.

Figure 1

Representation of the faces of the calcium oxalate crystals: (A) crystals COM; and (B) crystals COD. The numbers presented between parentheses represent each face of a crystal; the numbers displayed between brackets represent the direction of growth of each face of a crystal; and the numbers presented between braces represent the set of same faces of a same crystal.

Figure 2

(A) Electrophoretic mobility of the sulfated polysaccharides from C. cupressoides seaweed. Fifty micrograms of the Caulerpa SPs were applied on agarose gel prepared with 1,3-diaminopropane-acetate buffer 0.05 M, pH 9.0, and then subjected to electrophoresis at 90 V/cm for 60 min. The gel was maintained in 0.1% cetyltrimethylammonium bromide for 2 h, dried and subsequently stained with 0.1% toluidine blue (in 50% ethanol and 1% acetic acid in water) for 15 min. The gel was unstained with the same staining solution without the dye. (B) Gel permeation chromatography of sulfated polysaccharides from C. cupressoides. Two hundred micrograms of the Caulerpa SPs were applied to a Sephadex^® G-100 column (140 cm × 1 cm). The column was eluted with 0.2 M acetic acid, 1 mL fractions were collected, and the presence of sugars and sulfate was determinate as described in Methods Section.

Figure 3

Scanned Electron micrographs of the crystals formed morphotypes after incubation with polysaccharides of green seaweed C. cupressoides. The CaOx crystals were formed in a stable CaOx solution target (1 mM) in the absence (A,B) and in the presence of SPs C. cupressoides (0.25 mg/mL): (C,D) CCB-F0.3; (E,F) CCB-F0.5; and (G) CCB-F1.0; (H) CCB-F2.0. Black arrow shows the COD form; larp-arrow rounded head shows the COM shape; arrow tipped diamond shows the COT form. White arrow indicates tetragonal bipyramid structures with thicker (100) faces.

Figure 4

Infrared spectrum of COM crystals, COD crystals and CaOx crystals formed after incubation with Caulerpa SPs (CCB-F0.3 and CCB-F2.0). The values in red indicate the bands that are characteristic of the COM crystals; those in blue are characteristic of COD crystals; in green are the values that indicate the presence of sulfate; and in pink is the band found in the spectra of both crystals (COM and COD).

Figure 5

Fluorescent crystal analyzes by flow cytometry and fluorescence microscopy. (A) CaOx crystals were formed in the presence of fluorescently unlabeled SPs (0.1 mg/mL) and FITC (0.1 mg/mL) (blue graphics) or in the presence of fluorescent SPs (0.1 mg/mL) (pink graphics) and detected by flow cytometry. The number within each square corresponds to the percentage of crystals labeled fluorescently. (B) Comparison between crystals in light field microscopy (non-fluorescent), observed in the upper line, and the SPs-FITC-conjugated crystals (fluorescent in green), observed in the bottom line. FITC corresponds to CaOx crystals formed in the presence of FITC (0.1 mg/mL). These crystals were not fluorescent. The assays were performed twice (n = 3).

Figure 6

Crystal surface atomic composition characterization by EDS. (A) SEM image of CaOx with the presence of CCB-F0.5. The black squares (apex, face, and base) indicated the point where the EDS analyses were performed to quantify the sulfur atoms. (B) Percentage of sulfur distribution in different parts of CaOx and CaOx treated with polysaccharide. (C) Calcium (red dots) and oxygen (blue dots) marking in CaOx with the presence of CCB-F0.5. (D) Increase in sulfur marking (green dots) in CaOx with the presence of CCB-F0.5.