This study evaluated membrane stabilization and detoxification potential of ethyl acetate fraction of
The kidney is a highly specialized organ that maintains the body’s homeostasis by selectively excreting or retaining various substances according to specific body needs. In its role as a detoxifier and primary eliminator of xenobiotics, it becomes vulnerable to developing injuries. Such injuries have been linked with reactive oxygen species (ROS) mediated oxidative stress on renal biomolecules [
Besides very limited research on the therapeutic importance of CS in Africa, opinions on its nephroprotective potential are divergent. While Sukandar et al. [
Assay kits for kidney function parameters, glutathione peroxidase, and glutathione reductase were purchased from Randox Laboratories Limited, United Kingdom. Acetaminophen (APAP) and vitamin C were products of Emzor Pharmaceuticals, Lagos, Nigeria. The water used was glass-distilled and all other chemicals and reagents were of analytical grade.
Fresh corn silks were harvested from a maize plantation in the Phuthaditjhaba area of Maluti-A-Phofung, QwaQwa, Free State province, South Africa, between November 2014 and March 2015. They were authenticated by Dr. A. O. T. Ashafa of the Plant Sciences Department, University of the Free State, QwaQwa Campus, South Africa. Voucher specimen (number SabMed/01/2015/QHB) was thereafter prepared and deposited at the Herbarium of the University.
The CS was shade dried to constant weight and subsequently milled by an electric blender (model MS-223; Labcon PTY, Durban, South Africa) to fine powder. The powdered sample (2 kg) was extracted with 70% methanol (10 L) with regular agitation for 24 h. The solution obtained was filtered (Whatman no. 1 filter paper) and the resulting filtrate concentrated to a yield of 455 g crude extract. Part of the crude extract (400 g) was suspended in distilled water (0.6 L) and subsequently partitioned in succession with n-hexane, dichloromethane, ethyl acetate, and n-butanol. This yielded 18 g, 24 g, 33 g, and 42 g of the respective fractions. About 10
This study was approved (UFS-AED2015/0005) by the Ethical Committee of University of the Free State, South Africa, in accordance with the Guidelines of the National Research Council Guide for the Care and Use of Laboratory Animals [
This was achieved as previously described [
Fifty rats randomized into 9 experimental groups were used for this study. While the nephrotoxic control group had 10 animals that were further divided into 2 sets (with one of the sets designated as satellite group) to monitor possible self-recovery effects, the remaining animals (40) were evenly distributed into 8 treatment groups of 5 rats each and treated as in Table
Groups | Designation | Treatments |
---|---|---|
1 | Control | Given sterile placebo. |
2 | Nephrotoxic rats in two sets | Animals induced with nephrotoxicity and not treated. |
3 | CSEAF-treated only | Given 200 mgkg−1 b.w. of CSEAF only for 14 days. |
4, 5, and 6 | Pretreatment | Pretreated with CSEAF (100 and 200 mgkg−1 b.w.) and vitamin C (200 mgkg−1 b.w.), respectively, for 14 days prior to nephrotoxicity induction. |
7, 8, and 9 | Posttreatment | Nephrotoxic rats posttreated, respectively, with the fraction (100 and 200 mgkg−1 b.w.) and vitamin C (200 mgkg−1 b.w.) for 14 days. |
Treatments were done once daily via oral intubation between 9.00 and 10.00 a.m. to minimize possible diurnal effects. A transition period of 24 h was observed between the two subsequential treatment periods in both pre- and posttreatment groups.
Forty-eight hours after the last treatment in each case, the rats were humanely euthanized under halothane anaesthetization and blood was collected via cardiac puncture into plain sample bottles. For serum preparation, the blood was allowed to clot for 10 min and subsequently centrifuged (Beckman and Hirsch, Burlington, IO, USA) at 3,000 ×g for 15 minutes. Serum was carefully aspirated and used for kidney function tests. The rats were also immediately dissected and the kidneys were diligently harvested, blotted with clean tissue paper, cleaned of fat, and weighed and the relative kidney-body weight ratios (RKW) were evaluated. The left kidney was thereafter sliced into two portions with one of the portions homogenized in Tris-HCl buffer (0.05 mol/L Tris-HCl and 1.15% KCl, pH 7.4) for antioxidant analyses, while the other was used for histological examination.
Following the procedures outlined in the assay kits, kidney function parameters were determined. Serum concentrations of creatinine, blood urea nitrogen, uric acid, potassium, sodium, and calcium were evaluated. Creatinine clearance rate (CCR) was estimated as earlier reported [
The procedure described by Ellman [
For GSSG level estimation, the described method of Hissin and Hilf [
The homogenate levels of lipid peroxidation products (conjugated dienes, lipid hydroperoxides, and malondialdehyde) were estimated as reported by Reilly and Aust [
The method of Levine et al. [
For the AOPP assay, the kidney homogenate (2 mL) was centrifuged at 2500 ×g for 10 min at 40°C. The resulting supernatant was thereafter added to a reaction mixture containing 50% acetic acid and 1.16 mol/L potassium iodide in phosphate buffered saline solution. The absorbance was read at 340 nm and the concentration of AOPP determined from the extrapolated standard curve of serially diluted AOPP standard solution using 500
The quantity of fragmented DNA in the kidney homogenates was determined using standard protocols [
Homogenate activities of glutathione peroxidase (GPx) and glutathione reductase (GRx) were also evaluated as per the manufacturer’s instructions in the assay kits.
The activity of superoxide dismutase (SOD) in the tissue homogenate was determined as outlined by Misra and Fridovich [
The homogenate activity of catalase (CAT) activity was evaluated adopting the method of Aebi [
Following previously reported standard protocol [ normal and well-preserved renal architecture; proximal convoluted tubules dilatation, focal granulovacuolar epithelial cell degeneration, and granular debris in not more than 1% of the tubular lumen; epithelial necrosis and desquamation involving less than 50% of cortical tubules; epithelial desquamation and necrosis involving more than 50% of proximal tubules; complete or almost entire tubular necrosis.
This was achieved following the method of Oyedapo et al. [
Earlier reported methods [
Degrees of protection conferred on the DNA, kidney function parameters, and inhibition of hemolysis by the fraction were expressed as percentages. Other results were subjected to one-way analysis of variance (ANOVA) using SPSS software package for windows (Version 16, SPSS Inc., Chicago, USA) and presented as mean ± standard error of mean (SEM) of five determinations. Significant difference between the treatment means was determined at 95% confidence level using Duncan’s Multiple Range Test.
Data obtained with respect to body weight gain revealed significant (
Effect of
Treatments | Weight changes | Kidney weight (g) | RKW (g/100 g b.w.) | ||
---|---|---|---|---|---|
Initial (g) | Final (g) | % weight gain | |||
Sterile placebo (control) | 210.22 ± 0.90 | 224.01 ± 0.89 | 6.16 |
1.61 ± 0.01 |
0.72 |
APAP treatment | 220.12 ± 0.77 | 215.00 ± 0.99 | (2.34 |
0.97 ± 0.01 |
0.45 |
200 mg/kg b.w. CSEAF | 200.05 ± 0.54 | 220.01 ± 0.86 | 9.07 |
1.69 ± 0.01 |
0.77 |
100 mg/kg b.w. CSEAF, then APAP | 212.21 ± 0.75 | 223.09 ± 0.76 | 4.88 |
1.58 ± 0.02 |
0.71 |
200 mg/kg b.w. CSEAF, then APAP | 206.15 ± 0.45 | 227.31 ± 0.69 | 9.31 |
1.73 ± 0.01 |
0.76 |
200 mg/kg b.w. vitamin C, then APAP | 215.05 ± 0.39 | 228.19 ± 0.50 | 5.76 |
1.64 ± 0.02 |
0.72 |
APAP, then 100 mg/kg b.w. CSEAF | 200.15 ± 0.31 | 205.99 ± 0.56 | 2.84 |
1.49 ± 0.02 |
0.73 |
APAP, then 200 mg/kg b.w. CSEAF | 219.02 ± 0.97 | 232.00 ± 0.67 | 5.60 |
1.72 ± 0.01 |
0.74 |
APAP, then 200 mg/kg b.w. vitamin C | 221.09 ± 0.75 | 227.23 ± 0.45 | 2.70 |
1.63 ± 0.01 |
0.72 |
Values bearing different superscripts along the same column for each parameter are significantly different (
Parenthesis signifies reduced value for the parameter. RKW: relative kidney-body weight.
Significantly (
Effect of
Treatments | Creatinine (mg/dL) | BUN (mg/dL) | Uric acid (mg/dL) | CCR (mL/min) |
---|---|---|---|---|
Sterile placebo (control) | 0.63 ± 0.02 |
15.66 ± 0.17 |
4.09 ± 0.01 |
10.48 ± 0.01 |
APAP treatment | 2.50 ± 0.04 |
49.00 ± 0.26 |
15.99 ± 0.02 |
2.64 ± 0.01 |
200 mg/kg b.w. CSEAF | 0.62 ± 0.01 |
14.99 ± 0.43 |
4.10 ± 0.03 |
10.65 ± 0.02 |
100 mg/kg b.w. CSEAF, then APAP | 0.99 ± 0.03 |
22.11 ± 0.23 |
8.11 ± 0.09 |
6.67 ± 0.01 |
200 mg/kg b.w. CSEAF, then APAP | 0.57 ± 0.02 |
15.99 ± 0.13 |
4.51 ± 0.03 |
11.58 ± 0.02 |
200 mg/kg b.w. vitamin C, then APAP | 0.56 ± 0.02 |
16.00 ± 0.12 |
4.51 ± 0.09 |
11.79 ± 0.01 |
APAP, then 100 mg/kg b.w. CSEAF | 1.11 ± 0.03 |
25.12 ± 0.18 |
8.99 ± 0.06 |
5.95 ± 0.02 |
APAP, then 200 mg/kg b.w. CSEAF | 0.63 ± 0.05 |
15.79 ± 0.12 |
9.01 ± 0.06 |
10.48 ± 0.01 |
APAP, then 200 mg/kg b.w. vitamin C | 1.25 ± 0.01 |
16.09 ± 0.23 |
8.88 ± 0.04 |
5.28 ± 0.01 |
Effect of
Treatments | Sodium (mEq/L) | Potassium (mEq/L) | Calcium (mg/dL) |
---|---|---|---|
Sterile placebo (control) | 135.44 ± 1.35 |
3.50 ± 1.01 |
8.95 ± 1.01 |
APAP treatment | 338.22 ± 1.65 |
10.11 ± 1.02 |
3.33 ± 1.00 |
200 mg/kg b.w. CSEAF | 145.01 ± 1.21 |
3.59 ± 1.00 |
9.83 ± 1.02 |
100 mg/kg b.w. CSEAF, then APAP | 198.22 ± 1.11 |
5.55 ± 1.02 |
5.99 ± 1.01 |
200 mg/kg b.w. CSEAF, then APAP | 138.94 ± 1.26 |
3.75 ± 1.06 |
9.01 ± 1.00 |
200 mg/kg b.w. vitamin C, then APAP | 143.12 ± 1.70 |
3.55 ± 1.01 |
9.17 ± 1.01 |
APAP, then 100 mg/kg b.w. CSEAF | 226.21 ± 1.13 |
7.09 ± 1.01 |
6.99 ± 1.02 |
APAP, then 200 mg/kg b.w. CSEAF | 200.12 ± 1.42 |
5.99 ± 1.00 |
8.99 ± 1.02 |
APAP, then 200 mg/kg b.w. vitamin C | 139.01 ± 1.22 |
3.79 ± 1.02 |
9.15 ± 1.01 |
Mean percentage protection offered by
The effects of 14-day treatment with CSEAF on the nonenzymic antioxidant status and oxidative stress markers of the experimental rats are presented in Table
Effect of
Treatment | GSH (X) | GSSG (X) | GSH/GSSG | PC (X) | AOPP (Y) | F/DNA (%) |
---|---|---|---|---|---|---|
Sterile placebo (control) | 35.11 ± 1.35 |
0.20 ± 0.01 |
175.55 ± 0.20 |
3.72 ± 0.25 |
195.98 ± 1.99 |
10.12 ± 0.10 |
APAP treatment | 7.56 ± 1.09 |
1.99 ± 0.02 |
3.80 ± 0.08 |
15.12 ± 0.22 |
501.23 ± 1.59 |
65.15 ± 0.19 |
200 mg/kg b.w. CSEAF | 25.02 ± 1.08 |
0.12 ± 0.03 |
208.50 ± 0.32 |
3.66 ± 0.40 |
190.23 ± 1.45 |
11.75 ± 0.14 |
100 mg/kg b.w. CSEAF, then APAP | 30.19 ± 1.11 |
0.21 ± 0.02 |
143.29 ± 0.19 |
8.00 ± 0.25 |
275.99 ± 1.40 |
30.16 ± 0.15 |
200 mg/kg b.w. CSEAF, then APAP | 31.01 ± 1.06 |
0.16 ± 0.03 |
193.81 ± 0.15 |
4.67 ± 0.32 |
193.01 ± 1.34 |
12.01 ± 0.18 |
200 mg/kg b.w. vitamin C, then APAP | 36.11 ± 1.32 |
0.19 ± 0.02 |
190.05 ± 0.17 |
3.89 ± 0.25 |
200.45 ± 1.29 |
11.21 ± 0.19 |
APAP, then 100 mg/kg b.w. CSEAF | 15.99 ± 1.11 |
0.22 ± 0.01 |
72.68 ± 0.15 |
9.04 ± 0.22 |
287.32 ± 1.88 |
31.00 ± 0.10 |
APAP, then 200 mg/kg b.w. CSEAF | 16.21 ± 1.00 |
0.19 ± 0.05 |
85.32 ± 0.09 |
9.14 ± 0.35 |
203.19 ± 1.25 |
12.09 ± 0.11 |
APAP, then 200 mg/kg b.w. vitamin C | 15.15 ± 1.72 |
0.19 ± 0.05 |
79.74 ± 0.10 |
8.01 ± 0.29 |
212.34 ± 1.67 |
12.13 ± 0.15 |
Effect of
Kidney homogenate activities of GRx, GPx, SOD, and CAT were significantly (
Effect of
Treatments | Antioxidant enzymes (nmol min−1 mgprotein−1) | |||
---|---|---|---|---|
SOD | Catalase | Glutathione Rx | Glutathione Px | |
Sterile placebo (control) | 43.12 ± 0.15 |
33.99 ± 0.11 |
56.11 ± 0.45 |
123.15 ± 1.10 |
APAP treatment | 10.11 ± 0.10 |
13.29 ± 0.12 |
18.11 ± 0.23 |
43.66 ± 1.15 |
200 mg/kg b.w. CSEAF | 59.21 ± 0.03 |
45.35 ± 0.11 |
55.19 ± 0.20 |
144.11 ± 1.10 |
100 mg/kg b.w. CSEAF, then APAP | 32.12 ± 0.11 |
22.76 ± 0.15 |
30.21 ± 0.25 |
102.31 ± 1.10 |
200 mg/kg b.w. CSEAF, then APAP | 41.24 ± 0.12 |
32.99 ± 0.19 |
53.99 ± 0.23 |
125.03 ± 1.11 |
200 mg/kg b.w. vitamin C, then APAP | 42.07 ± 0.21 |
33.09 ± 0.11 |
56.04 ± 0.35 |
100.19 ± 1.09 |
APAP, then 100 mg/kg b.w. CSEAF | 32.11 ± 0.12 |
22.18 ± 0.13 |
31.14 ± 0.31 |
98.22 ± 1.15 |
APAP, then 200 mg/kg b.w. CSEAF | 31.99 ± 0.15 |
31.00 ± 0.13 |
55.09 ± 0.26 |
120.11 ± 1.10 |
APAP, then 200 mg/kg b.w. vitamin C | 30.98 ± 0.20 |
30.01 ± 0.12 |
33.04 ± 0.35 |
96.12 ± 1.12 |
Macroscopic examination of kidneys from the control group revealed that they were essentially normal with characteristic fine texture and dark maroon appearance. While kidneys from the fraction-administered animals showed mild spots of brown color changes, those of the APAP-intoxicated animals revealed color changes from maroon to brown with characteristic uneven texture. Detailed histoarchitectural examination of the kidney sections of the control and 200 mgkg−1 b.w. CSEAF groups showed no histological derangements, as evidenced by the normal and well-preserved renal architecture with characteristic intact glomeruli and tubules (Figures
Histopathological grading of liver tissue sections of
Treatments | Scores | ||||
---|---|---|---|---|---|
0 | 1 | 2 | 3 | 4 | |
Control | ( |
( |
( |
( |
( |
APAP treatment | ( |
( |
( |
( |
( |
200 mg/kg of CSEAF | ( |
( |
( |
( |
( |
100 mg/kg b.w. CSEAF, then APAP | ( |
( |
( |
( |
( |
200 mg/kg b.w. CSEAF, then APAP | ( |
( |
( |
( |
( |
200 mg/kg b.w. vitamin C, then APAP | ( |
( |
( |
( |
( |
APAP, then 100 mg/kg b.w. CSEAF | ( |
( |
( |
( |
( |
APAP, then 200 mg/kg b.w. CSEAF | ( |
( |
( |
( |
( |
APAP, then 200 mg/kg b.w. vitamin C | ( |
( |
( |
( |
( |
(
Kidney micrographs (×400, hematoxylin and eosin stained) of (a) control rat, (b) nephrotoxic rat, (c) CSEAF (200 mg/kg b.w.) treated rat, (d) nephrotoxic rat pretreated with CSEAF (100 mg/kg b.w.), (e) nephrotoxic rat pretreated with CSEAF (200 mg/kg b.w.), (f) nephrotoxic rat pretreated with vitamin C (200 mg/kg b.w.), (g) nephrotoxic rat posttreated with CSEAF (100 mg/kg b.w.), (h) nephrotoxic rat posttreated with CSEAF (200 mg/kg b.w.), and (i) nephrotoxic rat posttreated with vitamin C (200 mg/kg b.w.). CSEAF: corn silk ethyl acetate fraction, BC: Bowman’s capsule showing leukocyte infiltration, DT: dilated proximal tubule, and NA: necrotic area.
The result of membrane stabilizing activity of CSEAF is shown in Figure
Effect of
Acetaminophen-mediated oxidative nephrotoxicity has been well documented and is characterized by morphologic and functional evidence of proximal tubular injury in humans and experimental animals [
In this study, the increased serum concentrations of creatinine, blood urea nitrogen, and uric acid coupled with the attenuated CCR in the APAP-intoxicated animals may be indicative of renal injury and cell necrosis resulting from formation of NAPQI in excess of GSH detoxification ability. This is consistent with previous studies [
The most common cause of electrolyte imbalance or disturbance is associated with renal failure [
Changes in the body weight of animals have been used to predict the nature and extent of drug-induced toxicity and may give important information on their overall health status [
Studies have implicated NAPQI formation and oxidative stress in APAP-induced nephrotoxicity [
An elevated level of malondialdehyde in tissues is an obvious indication of cellular damage due to lipid peroxidation resulting from malfunctioning of the antioxidant defense system [
Similar to protein-oxidized products, calcium ion accumulation and hydroxyl radical mediated oxidative damage are important events in the pathogenesis of DNA fragmentation. These events promote either tissue necrosis or carcinogenesis, which subsequently results in cell death [
During renal damage, superoxide radicals are formed at the site of injury and if their accumulation exceeds the body’s antioxidant capacity it could overwhelm the defensive activities of SOD and CAT, thereby aggravating the severity of existing damage [
In addition to complementing biochemical analyses, histopathological examination of kidney sections may provide invaluable information on how pharmacologically potent an agent is against renal damage. The significant alterations in glomerular structure, the thickening of the glomerular basement membrane, widening of the filtration slits, basal infolding patterns with the presence of cytoplasmic vacuolation, and the increase in collagen deposition around the tubules as observed in the kidney sections of APAP-intoxicated rats could be responsible for their impaired renal function. The consequently decreased glomerular filtration rate with associated elevated serum levels of urea and creatinine was evident in the present study and agreed with a previous report [
During inflammatory events, lysosomal enzymes and hydrolytic constituents are released from phagocytes into the extracellular space. This release consequently inflicts injuries on the surrounding organelles and tissues as well as aggravating the severity of any existing infection [
The reduction of oxidative onslaughts posed by APAP via treatments with the ethyl acetate fraction of corn silk is a manifestation of its capabilities to preserve renal function and delay progression of renal pathological conditions to end stage disease/death. The engendered nephroprotective effect by the fraction could, in rats, be ascribed to its antioxidative and membrane stabilization potential. This is achieved by facilitating detoxification of APAP-mediated nephrotoxicity via induction of ROS detoxifying enzymes, thereby stalling autooxidation of cellular macromolecules and renal tubular damage. Though the effects were prominently exhibited in the fraction-pretreated groups, the overall effects elicited in both treatment groups were remarkable and indicative of an excellent candidate for the management of drug-induced renal oxidative disorders.
The authors declare that there are no competing interests.
While appreciating the provision of Community Development Action Research Grant (no. KWA-SUCCD/2014AR/1/32) by Kwara State University, Malete, Nigeria, and the supportive grant of Nigerian Tertiary Education Trust Fund (TETFund) to S. Sabiu to South Africa, the expertise enjoyed at the laboratory of Phytomedicine and Phytopharmacology Research Group, University of the Free State, QwaQwa Campus, South Africa is also gratefully acknowledged.