Fig 1.
DPPH dose-dependent radical scavenging activity of C. officinarum AE.
Results are the mean ± SD of more than 3 different experiments. The median of each sample differs from the others (p<0.05) as inferred by the Kruskal Wallis test followed by Dunn’s post test.
Fig 2.
Chromatogram of C. officinarum AE.
From the obtained chromatogram the major peak was identified as Chlorogenic Acid.
Fig 3.
Percentage of stimulation of nucleation produced by C. officinarum AE.
Results are the mean ± SD of more than 3 different experiments. The median of each sample differs from the others (p<0.05) as inferred by the Kruskal Wallis test followed by Dunn’s post test.
Fig 4.
Light micrographs of CaOx crystals grown in the absence and presence of C. officinarum AE increasing concentrations.
(A) and (B) Controls; (C) AE, 2 μg dw/ml; (D) AE, 30 μg dw/ml; (E) AE, 125 μg dw/ml; (F) AE, 200 μg dw/ml; (G) AE, 500 μg dw/ml; (H) AE, 1000 μg dw/ml. Magnification was 400x for all panels.
Fig 5.
XRPD diffraction diagrams of calcium oxalate monohydrate (COM) and dihydrate (COD) crystals.
The crystals were grown in the absence and presence of C. officinarum AE: (a) COM crystals obtained in absence of AE.; (b, c) COM and COD crystals obtained in the presence of AE 200 μg dw/ml (b) and 500 μg dw/ml (c); (d) COD crystals obtained in the presence of AE 1000 μg dw/ml. D values and indices of the main reflections are reported. The standard diffraction spectra of COM (upper grey bar, reference code 00-016-0379) and COD (lower grey bar, reference code 01-075-1314) crystals are also shown for comparison.
Fig 6.
Scanning electron micrographs of various morphological types of COM and COD obtained in the absence (a-d) or presence (e, f) of different concentrations of C. officinarum AE. (a) Twinned COM crystals viewed from a [1] direction. (b) Single and twinned COM crystals. (c) Double-twinned COM crystals viewed a [10] direction. (d) Super- and hyper-twinned COM crystals, forming flower-like aggregates. (e) Single tetragonal bipyramidal COD crystal in the presence of 500 μg dw/ml AE; (f) Single, tetragonal COD crystals and small penetration twins of flat COD crystals in the presence of 1000 μg dw/ml AE.
Fig 7.
Scanning electron micrographs (upper) and morphometric / numerousness data (lower) of the CaOx crystals obtained in the absence or presence of different concentrations of C. officinarum AE.
(A) Control; (B) 200 μg dw/ml; (C) 500 μg dw/ml.; (D) 1000 μg dw/ml of AE.
Fig 8.
Representative topographic images of CaOx crystals with increasing C. officinarum AE concentration.
(A) and (B) Control samples, (C) 1μg dw/ml AE, (D) 5μg dw/ml AE, (E) 10μg dw/ml AE, (F) 50μg dw/ml AE, (G) 100 μg dw/ml AE and (H) 1000μg dw/ml AE.
Fig 9.
Scatter plots of maximum height, average height and volume obtained by AFM imaging.
Dunnett’s multiple comparison test was employed to assess the differences. Only comparisons with control are reported. * P<0.05; *** P « 0.01.
Fig 10.
Scatter plots of phase data obtained by phase AFM imaging.
Dunnett’s multiple comparison test was employed to verify the statistical differences among the samples and only comparison with control are reported. *** P « 0.01.
Fig 11.
Surface pattern of three phase images by AFM.
The figure shows the probable effects of AE component(s) on the CaOx crystal surface. In contrast to control sample (A), in the presence of 50 μg dw/ml C. officinarum AE (B) small globular shapes are present increasing in size in the presence of 1000 μg dw/ml C. officinarum AE (C).
Fig 12.
Colour enhanced representative images of FF transformed amplitude signals.
Red arrows indicate the nanometric domains of the CaOx crystals free of AE adsorption. Is possible to observe a variation of surface structures dependent on C. officinarum AE increasing concentrations, that probably influence the crystal growth. (A) control sample, (B) 50 μg, (C) 100 μg and (D) 1000 μg dw/ml C. officinarum AE respectively. In (C) white arrows indicate probable AE component(s) adsorption.
Fig 13.
Light micrographs of CaOx crystals grown in the absence and presence of C. officinarum solubilized component(s) in increasing concentrations.
(A) and (B) Controls; (C) 1.2 μg dw/ml; (D) 2.3 μg dw/ml; (E) and (F) 4.7 μg dw/ml; (G) 9.4 μg dw/ml; (H) 18.7 μg dw/ml. Magnification was 400x for all panels.
Fig 14.
Schematic illustration of hypothetic model for morphological changes of CaOx crystals induced by increasing doses of C. officinarum AE.
The diagram is indicative of the progressive adhesion of active components from C. officinurum on crystal surfaces as a hypothetic trigger factor directing the crystal modifications from COM towards COD forms. (A): Control; (B-D) progressively increasing doses of C. officinurum AE.