# Litholytic Activities of Natural Bioactive Compounds and Their Mechanism Insights
## 4 Litholytic Activity of Phenolic Acids
### In Vitro Investigations  
#### 4.1.1 Chitosan  
Chitosan, a natural polymer of marine origin [37], has numerous biomedical applications [38; 39]. However, chitosan tends to accumulate in renal tissue and promotes increased calcium excretion as well as the formation of COM crystals [35; 40]. In this context, recent research conducted by [PERSON] et al. [35] evaluated, in vitro, the effect of chitosan conju  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Compounds** & **Methods** & **Key Results** & **References** \\ \hline \multirow{8}{*}{Caffeic acid} & \multicolumn{3}{c}{Regulated the altered biochemical parameters} \\  & Ethylene glycol induced kidney stones in rats & Reduced CaOx deposits in kidneys & \\  & Doses: 20 and 40 mg/kg for 8 weeks & Down-regulated the osteepontin gene & [33] \\  & & Up-regulated the prothrombin fragment 1, & \\  & & Tamm–Horsfall, and bikumin genes & \\ \cline{2-4}  & \multicolumn{3}{c}{CaOx crystallization in synthetic urine} \\  & Forty microlitres of sodium oxalate 0.1 M was & Decreased the number of monohydrate and dihydrate & \\  & added per milliliter of urine & forms of crystals & \\  & Doses: 0.03, 0.1, and 0.3 mg/mL & & \\ \hline \multirow{8}{*}{Ferulic acid} & \multicolumn{3}{c}{Regulated the altered biochemical parameters} \\  & Ethylene-glycol (0.75\%)-induced renal calculus & Inributed oxidative stress & \\  & in experimental rats & Increased GSH, SOD, GPx, and CAT levels & \\  & Doses: 40 and 80 mg/kg daily for 28 days & Decreased lipid peroxidation (LPO) & \\  & & Decreased serum and urine levels of stone and & \\  & & renal markers & \\ \hline Gallic acid and & Measurement of crystallization of calcium & \\  & oxalate by spectrophotometry in artificial urine & Decreased the formation of CaOx crystals & [35] \\  & Dose: 10 mg of gallic acid per g of chitosan & & \\ \hline \multirow{8}{*}{Rosmarinic acid} & \multicolumn{3}{c}{Decreased urinary secretions of creatinine, uric acid,} \\  & & Ca\({}^{2+}\), and oxalic acid & \\  & & Decreased the concentration of creatinine, uric acid, & \\  & Ethylene-glycol-induced kidney stones in rats & and urea & \\  & Doses: 50 and 100 mg/kg & Decreased the contents of renal tissue oxalic acid and & \\  & & calcium in kidneys & \\  & & Decreased the calcium oxalate deposits in the kidneys & \\  & & and the pathological change degrees & \\ \cline{2-4}  & \multicolumn{3}{c}{CaOx crystallization in synthetic urine} \\  & Forty microlitres of sodium oxalate 0.1 M was & Decreased the number of monohydrate and dihydrate & \\  & added per milliliter of urine & forms of crystals in urine & \\  & Doses: 0.03, 0.1, and 0.3 mg/mL & & \\ \hline \hline \end{tabular}
\end{table}
Table 3: Anti-urolithiasis effects of phenolic acids.  
gated to gallic acid for its litholytic activity on CaOx. The effect of gallic acid-chitosan on the formation of crystalline CaOx was investigated, in particular on the nucleation, growth, and aggregation phases. Using gallic acid-chitosan, two distinct phases were observed (nucleation/growth and aggregation), while a phase with a 10-fold increase in absorbance value was reported using chitosan alone (nucleation/growth) [35]. Successively, the polarization microscopy, at the end of the experiment, revealed a decrease in the number of crystals in the gallic acid-chitosan assay compared to chitosan alone with a dominance of COM (9.25 \(\pm\) 4.04 crystals per field vs. 94.5 \(\pm\) 14.35 crystals per field, respectively). This effect was related to the amine group of gallic acid that binds to chitosan, leading to a decrease in the charge of this biopolymer and consequently to a small interaction with the crystals [35]. Thus, the conjugate gallic acid-chitosan can be used as a chitosan substitute. However, further in vivo assays should be conducted.