2.1 Drugs and chemicals
Ethane-1,2-diol was purchased from Titan Biotech Limited Bhiwadi-301019, Rajasthan, India. Tamsulosin hydrochloride was purchased from Sun Pharmaceutical Ind. Ltd. Dewas, India. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea, creatinine, uric acid, bicarbonate, albumin, and total protein estimation kits and saponin were obtained from Sigma-Aldrich Co, St Louis, USA. Calcium, sodium, potassium, chloride, and glucose estimation kits were purchased from AGAPPE Diagnostics Switzerland GmbH. All other chemicals used in the experiment were of the highest grade commercially available.
2.2 Plant material
The leaves of Triclisia gilletii were collected from a location at Okeluse, Ose Local Government Area of Ondo State in May 2016. The plant was identified by Mr. Omomoh B.E., and a voucher specimen (IFE-17536) of the authenticated Triclisia gilletii leaves was deposited in the herbarium of the Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria. The leaves were separated for other extraneous matter and subjected to shade drying.
2.3 Preparation of aqueous methanolic leave extract of Triclisia gilletii (TGME)
The air-dried leaves were subjected to a coarse powder using a dry grinder. The powdered leaves were soaked in 80% methanol for 72 h and filtered using Whatman filter paper no. 1 to obtain the aqueous methanolic extract (TGME). The filtered extracts were concentrated in a rotary evaporator and further concentrated to dryness using freeze dryer. After drying, a dark brown aqueous methanolic extract (17.21% w/w) was obtained.
2.4 Preliminary phytochemical screening and quantitative estimation of phytoconstituents
Preliminary phytochemical screening [13] of TGME was carried out to detect the presence of phenolics, flavonoids, steroids, saponins, and tannins. The total phenolic content was determined spectrometrically [14] and expressed as milligram of gallic acid equivalents (GAEs) per gram of extract. Total flavonoid content was measured by aluminum chloride colorimetric assay [15] and expressed as milligram of quercetin equivalent per gram of extract. Total tannins content was measured according to the method described by Broadhurst et al. [16] and expressed as milligram of catechin equivalent per gram extract. Total saponins were determined according to the method of Brunner [17] and expressed as milligram saponin equivalent per gram sample.
2.5 Nucleation assay
The method as described by [18] was used to study oxalate crystallization. In this method, crystallization was studied without inhibitor (i.e., control) and with extract Triclisia gilletii (i.e., inhibitor). The result was expressed in percentage. The following reaction resulted in the growth of crystals [19]:
$$\begin{aligned} & {\text{CaCl}}_{2} + {\text{Na}}_{2} {\text{C}}_{2} {\text{O}}_{4} \to {\text{CaC}}_{2} {\text{O}}_{4} + 2{\text{NaCl}} \\ & \left( {{\text{Calcium chloride}} + {\text{Sodium oxalate}} \to {\text{Calcium oxalate}} + {\text{Sodium chloride}}} \right). \\ \end{aligned}$$
2.6 Animal studies
Sixty healthy male Wistar rats (age 6 weeks old; body weight 150–180 g) obtained from a private breeder in Akure metropolis of Ondo State, Nigeria, were used for the study. The animals were housed at standard housing condition (27 ± 3 °C) under 12-h light/dark cycle in polypropylene pathogen-free cages and fed standard rodent chow (Vita Feeds Nigeria Limited) and water ad libitum.
2.7 Acute toxicity study
Healthy Wistar rats, starved overnight, were subjected to acute toxicity studies to determine non-observable adverse effect dose level (NOAEL) by acute toxic class method [20]. The rats (n = 3) were orally administered TGME in the limit test dose of 5000 mg/kg orally and observed continuously for behavioral, neurological, and autonomic profiles for 2 h and after a period of 24 h for any lethality, moribund state, or death and approximate LD50 determination. Dosages between 0 and 1000 mg/kg were repeated every other day for 7 days. Animals were euthanized via cervical dislocation 24 h after the last administration, blood collected for serum biochemical analysis of AST, ALT, ALP activity, and urea, creatinine, and uric acid concentration using Randox diagnostic kits.
2.8 Grouping, treatments, and induction of urolithiasis
The rats (n = 6) were divided into 7 groups.
Group I Control—Animals in this group were allowed access to distilled water ad libitum and 1 ml/kg distilled water for 28 days.
Group II (Induced)—Animals in this group were allowed access to drinking water containing 1% ethane-1,2-diol ad libitum and 1 ml/kg distilled water for 28 days.
Group(s) III–V—Animals in these group(s) were allowed access to drinking water containing 1% ethane-1,2-diol ad libitum with co-administration of aqueous methanol leaf extract of Triclisia gilletii (50, 100, 200 mg/kg b.w.) orally for 28 days.
Group VI—Animals in this group were allowed access to drinking water containing 1% ethane-1,2-diol ad libitum with co-administration of tamsulosin hydrochloride (standard drug) orally for 28 days.
Group VII—Animals in this group were administered the highest dosage of the aqueous methanol leaf extract of Triclisia gilletii (200 mg/kg b.w.) orally for 28 days.
The groups II–VI received 1% ethane-1,2-diol in drinking water ad libitum for 28 days, respectively, to induce urolithiasis and generate CaOx deposition into kidneys. The experimental model of urolithiasis utilizes the toxic mechanism of ethane-1,2-diol poisoning. Following ingestion, ethane-1,2-diol is first hepatically metabolized to glycolaldehyde by alcohol dehydrogenase. Glycolaldehyde is then oxidized to glycolic acid, glyoxylic acid, and finally oxalic acid. Ethane-1,2-diol-induced urolithiasis model mimics the human clinical condition of lithiasis, whereby oxalate produced chelate calcium ions forming insoluble CaOx and ultimately leading to nephrotoxicity and renal failure [2].
2.9 General observations
During the study period, body weight, water intake, and animal health was observed regularly, so that stressed and unhealthy animals were excluded from the study.
2.10 Collection and analysis of urine
All animals were kept in individual metabolic cages, and 24-h urine samples were collected on 0, 7, 14, 21, and 28th day of calculi induction treatment for the assessment of volume and urinalysis using urinalysis reagent strips (ACON Laboratories Inc. San Diego, USA). Urine collected on the 28th day was assessed for creatinine, urea, uric acid, electrolytes (calcium, potassium, sodium, chloride, and bicarbonate), glucose, albumin, and total protein concentrations following methods in recommended kits.
2.11 Serum analysis
Blood was collected via cardiac puncture after euthanizing with cervical dislocation; serum was separated by centrifugation at 10,000 g for 10 min and analyzed for creatinine, urea, uric acid, electrolytes (calcium, potassium, sodium, chloride, and bicarbonate), glucose, albumin, and total protein concentrations using methods recommended in the kits.
2.12 Analysis of kidney homogenate sample
The abdomen was cut open to remove both kidneys from each animal. Isolated kidneys were cleaned off extraneous tissue and rinsed in ice-cold physiological saline. The kidney was finely minced, and 20% homogenate was prepared in Tris–HCl buffer (0.02 mol/L, pH 7.4). Total kidney homogenate was used for assaying antioxidant parameters such as lipid peroxidation [21], glutathione peroxidase (GPx) activity [22], superoxide dismutase (SOD) activity [23], estimation of reduced glutathione (GSH) level [24], protein carbonyl concentration [25], ferric reducing antioxidant power [26], anti-inflammation—myeloperoxidase activity [27], markers of kidney mitochondria integrity—complex I activity [28], glutamine synthetase activity [29], Na+K+ ATPase activity [30], lactate dehydrogenase (LDH) activity [31], and protein content [32].
2.13 Histopathological analysis
Histopathological analysis was carried out as described by Sikarwar et al. [2]. The slides were examined under a light microscope to study the light microscopic architecture of the kidney and calcium oxalate deposits using H and E (hematoxylin and eosin) staining.
2.14 Statistical analysis
The data were analyzed with one-way ANOVA followed by Duncan multiple comparison post hoc tests. A statistical difference of p < 0.05 was considered significant in all cases.