# Litholytic Activities of Natural Bioactive Compounds and Their Mechanism Insights
## 5 Litholytic Activity of Other Compounds
### In Vitro Investigations  
Several studies [43; 44; 45; 46; 47; 48] have reported, _in vitro_, the effect of hydroxyanthraquinones, sulfated polysaccharides, L-arginine, vitamin E, DADS, DATS, betulin, and piper on the prevention of nephrolithiasis formation using the crystallization process in human urine, titrimetric, conductometric titration, and crystal development in cell culture assays. [PERSON] and colleagues investigated, in vitro, the anti-lithiasis effect of five hydroxyanthraquinones on CaOX crystal formation induced by sodium oxalate and calcium chloride using conductometric titration. 1,2,5,8-tetrahydrooxy-9,10-anthraquinone and its glycosides inhibited the formation of CaOX more than other compounds (\(\Delta\)EP = 180 \(\mu\)L, \(\Delta\)EP = 170 \(\mu\)L, respectively) [43]. The author reported that the increase in the number of hydroxyl groups in the aglycone is the main factor responsible for good complexing properties. The _in vitro_ assay of four sulfated polysaccharides (fucoglucoxyglucuronan, heterofucan, and two glucans from the brown seaweed _Dicytopteris justii_) on CaOX crystallization assays were carried out by [PERSON] et al. [44]. Heterofucan and glucan (1) inhibited the nucleation process with 73.33% and 84.07%, respectively, while the inhibition of glucan (1) aggregation was greater than that of sodium citrate (86% vs. 84%). From this data, the authors suggested that glucans stabilize COD crystals and prevent them from turning into COM ones, which protects against violithiasis. Furthermore, the zeta potential (\(\zeta\)) of heterofucan was significantly reduced compared to the CaOX samples (+2.00 \(\pm\) 0.45 vs. +8.39 \(\pm\) 1.79 mV, respectively) due to the greater negative charge compared to other sulfate polysaccharides [44]. In other work carried out by [PERSON] et al. [45], L-arginine, at different concentrations (0.5, 0.1, and 0.05 ng/mL) was evaluated, in vitro, on renal cell lines from pigs and dogs (MDCK I, MDCK II, and LLC-PK1) induced by sodium oxalate in the concentration of 8 mM. Pretreatment with L-arginine significantly increased the survival of all cells in a dose-dependent manner compared to oxalate-treated cells. At 0.05 ng/mL, L-arginine showed a significant survival in MDCK I and MDCK II cell lines compared to oxalate treatment cells, but not for LLC-PK1 line (66% vs. 55%, 61% vs. 51%, and 42% vs. 32%, respectively). Vitamin E, at different concentrations (25, 15, and 5 \(\upmu\)M), has been investigated in vitro using the same experimental approach [45]. At 5 and 15 \(\upmu\)M, vitamin E pretreatment significantly improved the survival of all cells compared to oxalate-alone-treated cells. L-arginine showed a significant survival in MDCK I and MDCK II cell lines, at 5 \(\upmu\)M, compared to oxalate treatment cells, but not for the LLC-PK1 line (78% vs. 66%, 50% vs. 38%, and 58% vs. 34%, respectively). [PERSON] and colleagues indicated that vitamin E has the same effect on epithelial cell lines in different species, while L-arginine has different effects depending on the origin of the cell species [45]. Therefore, further investigations are necessary to determine the effect of these two compounds on human kidney epithelial cells and determine their mechanism of action. The anti-crystallization activity of taraxasterol, at different concentrations (2.5, 5, 7.5, and 12.5 \(\upmu\)g/mL) on CaOX induced by sodium oxalate in synthetic urine, has been investigated by [49]. Taraxasterol inhibited the nucleation process in a dose-dependent manner (26-64%). In addition, at 12.5 \(\upmu\)g/mL, this compound increased the number of COD crystals (52 vs. 25 for 10-15 \(\upmu\)m size of COD crystals) and decreased COM crystals (125 vs. 195 for 10-15 \(\upmu\)m size of COD crystals) compared to the control sample. Furthermore, taraxasterol decreased the size of COD crystals in a dose-dependent manner. [PERSON] et al.