Antioxidant and Polyphenol-Rich Ethanolic Extract of Rubia tinctorum L. Prevents Urolithiasis in an Ethylene Glycol Experimental Model in Rats

Abstract

Treatment of kidney stones is based on symptomatic medications which are associated with side effects such as gastrointestinal symptoms (e.g., nausea, vomiting) and hepatotoxicity. The search for effective plant extracts without the above side effects has demonstrated the involvement of antioxidants in the treatment of kidney stones. A local survey in Morocco has previously revealed the frequent use of Rubia tinctorum L. (RT) for the treatment of kidney stones. In this study, we first explored whether RT ethanolic (E-RT) and ethyl acetate (EA-RT) extracts of Rubia tinctorum L. could prevent the occurrence of urolithiasis in an experimental 0.75% ethylene glycol (EG) and 2% ammonium chloride (AC)-induced rat model. Secondly, we determined the potential antioxidant potency as well as the polyphenol composition of these extracts. An EG/AC regimen for 10 days induced the formation of bipyramid-shaped calcium oxalate crystals in the urine. Concomitantly, serum and urinary creatinine, urea, uric acid, phosphorus, calcium, sodium, potassium, and chloride were altered. The co-administration of both RT extracts prevented alterations in all these parameters. In the EG/AC-induced rat model, the antioxidants- and polyphenols-rich E-RT and EA-RT extracts significantly reduced the presence of calcium oxalate in the urine, and prevented serum and urinary biochemical alterations together with kidney tissue damage associated with urolithiasis. Moreover, we demonstrated that the beneficial preventive effects of E-RT co-administration were more pronounced than those obtained with EA-RT. The superiority of E-RT was associated with its more potent antioxidant effect, due to its high content in polyphenols.

Keywords: Rubia tinctorum L.; antioxidants; ethylene glycol; histophatology; polyphenols; urolithiasis.

Figure 1

Cumulative body weight difference (mean ± SEM) in the 6 groups (6 rats/group) over the ten days of the treatment. Vehicle control (G1): received distilled water; Lithiasic control (G2): received 0.75% EG + 2% AC; E-RT 1 g/kg (G3): received concomitantly E-RT (1 g/kg) plus 0.75% EG + 2% AC; E-RT 2 g/kg (G4): received concomitantly E-RT (2 g/kg) plus 0.75% EG + 2% AC; EA-RT 1 g/kg (G5): received concomitantly EA-RT (1 g/kg) plus 0.75% EG + 2% AC; EA-RT 2 g/kg (G6): received concomitantly EA-RT (2 g/kg) plus 0.75% EG + 2% AC.

Figure 2

Representative microscopic images of hematoxylin-and-eosin-stained kidney sections observed under a light microscope from rats of: the vehicle control (G1), the lithiasic control (G2), treated with E-RT at 1 g/kg (G3) and 2 g/kg (G4), treated with EA-RT at 1 g/kg (G5) and 2 g/kg (G6). Gm: Glomeruli, T: Tubular, DG: Dilatation of glomeruli, DT: Dilatation of tubular, KH: kidney hemorrhage.

Figure 3

Evaluation of antioxidant activity in kidney homogenates using: (A) lipid peroxidation determination (MDA) and (B) catalase (CAT) enzyme activities. G1: Vehicle control; G2: Lithiasic control; G3: E-RT 1 g/kg; G4: E-RT 2 g/kg; G5: EA-RT 1 g/kg; G6: EA-RT 2 g/kg. The values shown are mean ± SEM of animals from each group (n = 6). ### p ≤ 0.001 vs. lithiasic control.

Figure 4

General experimental design diagram.