Studies on the in vitro and in vivo antiurolithic activity of Holarrhena antidysenterica

Abstract

Holarrhena antidysenterica has a traditional use in the treatment of urolithiasis, therefore, its crude extract has been investigated for possible antiurolithic effect. The crude aqueous-methanolic extract of Holarrhena antidysenterica (Ha.Cr) was studied using the in vitro and in vivo methods. In the in vitro experiments, Ha.Cr demonstrated a concentration-dependent (0.25-4 mg/ml) inhibitory effect on the slope of aggregation. It decreased the size of crystals and transformed the calcium oxalate monohydrate (COM) to calcium oxalate dehydrate (COD) crystals, in calcium oxalate metastable solutions. It also showed concentration-dependent antioxidant effect against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and lipid peroxidation induced in rat kidney tissue homogenate. Ha.Cr (0.3 mg/ml) reduced (p < 0.05) the cell toxicity and LDH release in renal epithelial cells (MDCK) exposed to oxalate (0.5 mM) and COM (66 μg/cm(2)) crystals. In male Wistar rats, receiving 0.75 % ethylene glycol (EG) for 21 days along with 1 % ammonium chloride (AC) in drinking water, Ha.Cr treatment (30-100 mg/kg) prevented the toxic changes caused by lithogenic agents; EG and AC, like loss of body weight, polyurea, oxaluria, raised serum urea and creatinine levels and crystal deposition in kidneys compared to their respective controls. These data indicate that Holarrhena antidysenterica possesses antiurolithic activity, possibly mediated through the inhibition of CaOx crystal aggregation, antioxidant and renal epithelial cell protective activities and may provide base for designing future studies to establish its efficacy and safety for clinical use.

Fig. 1. Calcium Oxalate crystallization study

Effect of Holarrhena antidysenterica (Ha.Cr) and Potassium Citrate (k-Cit) on calcium oxalate crystallization. (A) and (B) are the typical tracing of the control and in the presence of Ha.Cr and potassium citrate. Panel (C) is concentration response curves of Holarrhena antidysenterica and potassium citrate on S_A of the turbidity curves, while (D) shows the % inhibition on the S_N. Symbols shown are mean ± S.E.M. (n = 3). S_N and S_A represent slope of nucleation and slop of aggregation respectively.

Fig. 2. Calcium Oxalate incubation study

Representative photographs, under inverted microscope (200x), of CaOx crystals developed in the metastable solutions in the absence (A) and in the presence of Holarrhena antidysenterica (Ha.Cr) 2 mg/ml (B) and 2 mM K-Citrate (C).

Fig. 3. Antioxidant activity

Concentration response curves of the free radical scavenging activity of the butylated hydroxytoluene (BHT) and Holarrhena antidysenterica (Ha.Cr), while bar-chart (B) representing lipid peroxidation inhibitory activity of two different concentrations of Ha.Cr and BHT. Inhibition is measure as % of the respective control experiments. The values shown are mean ± SEM (n=3).

Fig. 4. Effect on MDCK cells

Effects of various concentrations of Ha.Cr on MDCK cell survival in acclimatization media (A). B and C show the protective effect of Ha.Cr after exposure to 0.5mM oxalate or 66 mg/cm² COM respectively. While (D) and (E) shows the percent increase in LDH release against control by MDCK cells exposed Ox. (0.5 mM) and COM (66 μg/cm²) for 24 hours. Data shown are mean ± SEM of two separate experiments with 3 independent replicates. *P < 0.05, **P < 0.05 and ***P < 0.001

Fig. 5. Images of Crystalluria

Images of calcium oxalate crystals in 3 hrs morning urine collected from Normal control (A), Lithogenic Control (B) and treated with Holarrhena antidysenterica (Ha.Cr) (C), under light microscope at 400x magnification.

Fig. 6. Microscopic images of Kidney sections in the in vivo study

Representative microscopic images of the H and E stain of the kidney sections from normal (A), Lithogenic group (B) and Treated (C) with Ha.Cr. A1, B1 and C1 show the Pizzolato’s staining of the respective kidney sections.

Fig. 7. CaOx crystal deposition score in preventive study;

Calcium oxalate crystal deposition score after treatment with 0.75% EG, 1% NH₄Cl (lithogenic group); Ha.Cr 30 mg/kg and 100 mg/kg. Severity grade were assigned as 0 = < 1 crystals, 1 = 1–10, 2 = 11–30, 3 = 31–50, 4 = 51–75 and 5 = > 75 crystals; data are expressed as mean ± SEM. *p < 0.05, **p < 0.05 and ***p < 0.001