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Usunobun U, Okpiabhele A. Telfairia occidentalis Hook f. mitigates Carbon Tetrachloride induced Nephrotoxicity in Rat. Journal of Research in Applied and Basic Medical Sciences 2023; 9 (3) :130-137
URL: http://ijrabms.umsu.ac.ir/article-1-246-en.html
Department of Biochemistry, Faculty of Basic Medical Sciences, Edo State University Uzairue, Edo State, Nigeria , usunobun.usunomena@edouniversity.edu.ng
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Plants have played very important therapeutic roles in maintaining and enhancing the quality of human health for thousands of years. For decades now, natural compounds have continued to be considered as one of the promising therapeutic agents against cancer, cardiovascular diseases, aging, diabetes, and neurodegenerative disorders due to their wide variety of modes of action, efficiency, accuracy, and fewer side effects (1-2). Telfairia occidentalis Hook f., commonly called fluted pumpkin or ugwu and occurs in the forest zone of West and Central Africa most especially in Nigeria, Benin, and Cameroun, is well grown as a leaf vegetable (3-4). Telfairia occidentalis, darkish-green leafy Vegetable of the Cucurbitaceae family is used in herbal preparations for the management of many diseases such as anaemia, hypertension, diabetes, and heart diseases in Nigeria (5-6). Telfairia occidentalis is rich in minerals such as iron, potassium, phosphorus, calcium, and magnesium (7), as well as in antioxidants and phytochemical compounds such as phenols, cucubitacine, anthocyanins, flavonoids, tannins, β-carotene, lycopene, vitamins A, C, and E (8-9). It is a popular medicinal plant known locally for its antidiabetic, antiplasmodial, hypoglycemic, hypolipidemic and antibacterial properties with most of these benefits being reported in various parts and different preparations of the plant in the laboratories (10-13). Several extracts of T. occidntalis leaves have been reported to elevate antioxidants levels both in vivo and in vitro, and scavenge, prevent, or suppress the production of free radicals (14-16).

Materials & Methods
Silymarin, Hydrogen peroxide, KMnO4, Epinephrine, thiobabituric acid, carbon tetrachloride, and absolute ethanol (99.8%) were purchased from Sigma-Aldrich (USA). Biochemical assays kits were obtained from Randox Diagnostics (Randox, United Kingdom). All other chemicals and reagents were of analytical grade.
Collection and Extraction of Telfairia occidentalis Leaves:
Fresh leaves of T. occidentalis were purchased from Ekiosa market, Benin City, Edo State, Nigeria and identified by a taxonomist. The Fresh leaves were thoroughly rinsed and air-dried at room temperature (24ºC) and then pulverized, crushed into fine powder using a manual blender and weighed. Aqueous extract of the plants was prepared by soaking 1000 g of the dry powdered plant materials in 5 liters of double distilled water and then kept at room temperature for 48 hours (for thorough extraction). At the end of the 48 hours, the extract was filtered first through a Whatmann filter paper No. 42 (125 mm) and then through cotton wool. The filtrate was concentrated using a rotary evaporator with the water bath set 40ºC until the crude extract was obtained. The dried residue (crude extract) was then stored at 4ºC. Aliquot portions of the crude plant extract residue was weighed and dissolved in normal saline for use on each day of the experiments.
Experimental Design/Procedure:
Adult male albino rats were purchased and allowed to acclimatize for 7 days and maintained under standard conditions, provided pelleted growers’ mash (containing 18 % crude protein and 2600Kcal/kg metabolizable energy, Guinea Feed, Nigeria PLC) and drinking water ad libitum. A daily cycle of 12 hours of light and 12 hours of darkness were provided for the animals. The study was conducted on 40 healthy Wistar male albino rats weighing 190-200 g, randomly assigned to five treatment groups of 8 rats each. The study was carried out in accordance with the guidelines for ethical conduct in the care and use of nonhuman animals in research (17).
One group received distilled water and serve as normal control. Second group received carbon tetrachloride (CCl4) alone for 4 days. Third and fourth groups received CCl4 for 4 days prior to treatment with 200 mg/kg and 400 mg/kg T. occidentalis aqueous extract for 6 days, respectively. The last group received CCl4 for 4 days prior to treatment with Silymarin (100 mg/kg). With exception of normal control rats, all rats received a mixture of freshly prepared CCl44 in olive oil (1 ml/kg, 1:1 intraperitoneally) for 4 days. T. occidentalis at a dose of 200 mg/kg and 400 mg/kg was chosen based on the previous studies of Saalu et al. (18) and Akang et al. (19).
24 hours after last administration, the rats from each group was sacrificed by cervical dislocation and blood samples obtained through heart puncture via a syringe into sample bottles containing no anticoagulant. The blood samples collected in sample bottles were allowed to clot and subsequently centrifuged at 5000 rpm for 20 min at room temperature to obtain serum for biochemical assays. 
Biochemical Parameters:
Serum urea was determined using the RANDOX Kit according to the manufacturer’s instructions following the method of 20. Fawcett et al. (20), while serum creatinine was determined by using the Jaffe’s method. The determination of serum Total Cholesterol (TC) was by method of Searcy et al. (21) while serum triglyceride (TG) was by method of Tiez et al. (22).
Histopathological Analysis:
Immediately after sacrifice, the kidney of both the test and control rats were excised, dried with blotting paper, weighed and a portion instantly fixed in 10% phosphate buffered formalin. Fixed tissue samples were embedded in paraffin blocks and sections of 5 mm were prepared. Sections were stained with hematoxylin and eosin (H & E), and examined under Olympus/3H light microscope. Photomicrographs of the kidney were captured using a Moticam Images Plus 2.0 digital fitted to the light microscope.
Statistical Analysis:
Data obtained from this study were expressed as mean value ± standard deviation. Differences between means of groups were determined by One-way ANOVA using Statistical Package for social scientist (SPSS) v.20. The mean differences were compared with the Duncan multiple range test. A probability level of less than 5% (P˂0.05) was considered significant.
Kidney functional markers in the serum of control and treated rats are provided in Table 1. The result of kidney assessment showed that compared to control and extract treated groups, CCl4 alone rats exhibited a significant increase in serum Creatinine and Urea. However, treatment of CCl4-induced rats with T. occidentalis aqueous extract at a dose of 200 mg/kg and 400 mg/kg resulted in a significant reduction in Creatinine and Urea.

Table 1. Effects of Telfairia occidentalis aqueous leaf extract on Kidney function parameters in Carbon tetrachloride (CCl4) -induced wistar rats
Treatment groups Creatinine (mg/dl) Urea (mg/dl)
Control 0.61a±0.05 15.21a±1.17
CCl4 1.98b±0.11 65.87b±2.02
T. occidentalis (200 mg/kg) + CCl4 1.09c±0.09 31.43c±2.01
T. occidentalis (400 mg/kg) + CCl4 1.03c±0.05 24.63d±2.30
Silymarin  (100 mg/kg) + CCl4 0.99c±0.07 24.98d±2.51
Values are expressed as Mean ± Standard Deviation. Values with different superscripts down the column differ significantly (p<0.05).
The results presented in Table 2 showed the data obtained from the effects of T. occidentalis aqueous leaf extract on lipid profile in CCl4-induced toxicity in rats. The result showed that CCl4 induction elevated serum total cholesterol and triglyceride levels when compared to control and extract treated groups. However, it was also revealed that treatment with T. occidentalis produced a dose-dependent significant (p˂0.05) reduction in serum total cholesterol and triglycerides levels compared to the CCl4 alone group.
Table 2. Effects of Telfairia occidentalis aqueous leaf extract on Lipid Profile in Carbon tetrachloride (CCl4)-induced wistar rats
Treatment groups Tot Cholesterol (mg/dL) Triglyceride (mg/dL)
Control 98.38a±2.67 121.83±3.05
CCl4 176.26b±3.06 201.87±3.02
T. occidentalis (200 mg/kg) + CCl4 114.42c±3.02 140.01±2.31
T. occidentalis (400 mg/kg) + CCl4
Silymarin  (100 mg/kg) + CCl4
Values are expressed as Mean ± Standard Deviation. Values with different superscripts down the column differ significantly (p<0.05).
The result of histopathological analysis showed control rat kidney to express intact basement membranes, normal tubules, and normal capillary tufts (Figure 1A), while the kidney of rat that received CCl4 alone showed renal tubular nephrosis, discrete necrosis of some tubular cells, blood vessel congestion, severe vacuolar degenerated glomerulus, and swelling of several renal epithelium (Figure 1B). The photomicrograph of kidney of rat given CCl4 and 200 mg/kg T. occidentalis showed minimized necrosis in renal tubules (Figure 1C), whereas photomicrograph of kidney of rat given CCl4 and 400 mg/kg T. occidentalis had no observed necrosis expression (Figure 1D). Meanwhile photomicrograph of kidney of rat given CCl4 and 100 mg/kg Silymarin showing mild mild renal tissue vascular congestion (Figure 1E).

Fig.1. A: Photomicrograph of rat kidney showing intact basement membranes, normal tubules and normal capillary tufts. B: Photomicrograph of rat kidney given CCl4 alone showing renal tubular nephrosis, discrete necrosis of some tubular cells and blood vessel congestion, severe vacuolar degenerated glomerulus and swelling of several renal epithelia. C: Photomicrograph of rat kidney given CCl4 and 200 mg/kg T. occidentalis showing minimized necrosis in renal tubules. D: Photomicrograph of rat kidney given CCl4 and 400 mg/kg T. occidentalis with no observed necrosis expression. E: Photomicrograph of rat kidney given CCl4 and 100 mg/kg Silymarin showing mild mild renal tissue vascular congestion.
In assessment of the kidney functionality, serum urea elevation is an indication of kidney failure which normally degenerates to severe kidney damage in prolonged cases (23-24). More so, renal failure is believed to be linked to high serum levels of creatinine, which is a useful index in the diagnosis of chronic kidney disease. Creatinine is not reabsorbed at the Loop of Henle like urea, thus, this marker helps to understand the glomerular filtration rate of the kidney (25). The results of this study showed that CCl4 induction significantly increased urea and creatinine levels, thus indicating that the impact of CCl4 metabolism triggers glomerular cell and kidney tubule toxic­ity, similar to results of previous studies (26-29). The increased levels of blood urea and creatinine in CCl4-induced rats implies inability of the kidneys to excrete these by-products leading to their elevated levels in the blood and decreased excretion in urine (30). However, T. occidentalis at a dose of 200 mg/kg and 400 mg/kg similar to Silymarin at 100 mg/kg showed a decrease in urea and creatinine levels compared to CCl4 alone rats, thus restoring the abnormalities in the levels of these biomarkers with 400 mg/kg T. occidentalis extracts, having higher activity and bringing the kidney parameters close to normal, which also compared well with standard drug, silymarin similar to related findings of Usunobun et al. (28) and Okolie et al. (29). The renal maintenance following T. occidentalis treatment can be attributed to the phytoconstituents in T. occidentalis including flavonoids (9), which protected the kidney from free radical attacks and thus against oxidative stress and damage compared to the group that received CCl4 alone.
Lipids are known to play an important role in the incidence of liver disease. Increased levels of cholesterol and triglycerides are known to be associated with atherosclerosis and coronary heart disease (31). In this study, administration of CCl4 resulted in significant increase in total cholesterol and triglycerides in CCl4-alone rats compared to the control rats and T. occidentalis treated rats, a result that was found similar to the work of Sandhya et al. (32).  Thus, this study shows that CCl4 treatment causes a disruption of lipid metabolism as seen in the increased total cholesterol and triglyceride. The increase in total cholesterol and triglycerides can be attributed to free radicals generated from CCl4 metabolism and toxicity which damage the endoplasmic reticulum, leading to reduced protein synthesis and lipid accumulation in the liver (33-34). However, although in a higher dose dependent manner, administration of T. occidentalis at doses of 200 mg/kg and 400 mg/kg significantly lowered (p < 0.05) the levels of total cholesterol and triacylglycerol in comparison with CCl4 alone administered rats. The result of our study on the ability of T. occidentalis to lower CCl4 elevated blood lipids agrees with similar reported works (35-38). The ability to lower the lipid levels can be attributed to our previously reported phytoconstituents in T. occidentalis (9). Specifically, flavonoids have been reported to lower lipid levels by inhibiting lipid absorption, lipogenesis, and stimulating lipolysis (39).
The observed lesions seen in the photomicrograph of kidney of rats that received CCl4 and the nephroprotection observed following administration of T. occidentalis corroborated the results of the observed biochemical parameters.

In conclusion, the results of our study showed that CCl4 induction caused hepatorenal dysfunction characterized by altered biochemical, compromised cellular and structural integrity. However, treatment with aqueous leaf extract of T. occidentalis significantly reversed these anomalies, suggesting its protective effect against CCl4-induced hepatorenal damage. It can be exerted that T. occidentalis nephroprotective effect is associated with its ability to improve renal function parameters as it may have increased glomerular filtration rate, resulting in less serum creatinine and urea levels. The antioxidant properties and detoxification capacity of T. occidentalis may also be responsible for its protective effect against CCl4 toxicity.

Financial Support
This research was funded by Tertiary Education Trust Fund (TETFUND), Nigeria through the 2020 Institution Based Research (IBR) Grant.

The authors humbly appreciate the University management and the animal house staff of Edo State University Uzairue as well as TETFUND, Nigeria for all their support in this research.

Conflict of interest
We don’t have any conflict of interest.
Type of Study: orginal article | Subject: General

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