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1,2Maharaja Agrasen School of Pharmacy, Maharaja Agrasen University, Baddi H.P., India
2Faculty of Pharmacy, Uttar Pradesh University of Medical Science Saifai, U.P., India
The purpose of this study is to evaluate nephroprotective potential of Trichosanthes dioica Roxb. leaves extract against Cisplatin induced nephrotoxicity and renal dysfunction. Forty-two Wistar rats were selected for the study. Urea and creatinine in plasma, kidney weight, urine output, blood urea nitrogen, urinary sodium and potassium level, renal enzymatic and non-enzymatic antioxidants and lipid peroxidation were evaluated along with histopathological investigation in various experimental groups of rats. Trichosanthes dioica leaves extract 200 and 400 mg/kg treatment to Cisplatin treated rats recorded significant decrement (up to p < 0.01) urea and creatinine in plasma, urinary Na+ and K+ level, renal lipid peroxidation along with significant increment (up to p < 0.01) in renal enzymatic and non-enzymatic antioxidants. Histological observations of kidney tissues too were correlated with the biochemical observations. These finding powerfully supports that Trichosanthes dioica leaves extract acts in the kidney as a potent scavenger of free radicals to prevent the toxic effects of Cisplatin both in the biochemical and histopathological parameters.
Trichosanthes dioica R. is an important medicinal herb and in Charak Samhita, leaves and fruits of this plant are used for treatment of alcoholism, jaundice, oedema and alopecia. Over 20 species of Trichosanthes are recorded in Asia, among those T. dioica are cultivated as vegetable. Trichosanthes dioica (Pointed gourd) is known by the name of parwal, parmal, patol, potala in different parts of India and Bangladesh and used as antipyretic, diuretic, cardiotonic and laxative [1,2,3]. The fruit and leaves are the edible part of the plant which are cooked in various ways either alone or in combination with other vegetables. Juice of leaves of T. dioica is used as tonic, febrifuge and in subacute cases of enlarged liver and spleen. The various chemical constituents present in T. dioica are vitamin A, vitamin C, tannins, saponins [4]. Aqueous extract of T. dioica fruits showed the anti-diabetic activity, cholesterol lowering activity in normal and streptozotocin induced diabetic rats, hepatoprotective activity against ferrous sulphate-induced liver injury and skin disorder. The fixed oil of seeds of Trichosanthes species including T. dioica have antifungal property [5,6]. To the best of our knowledge, there were no any scientific reports available in support of its nephroprotective potential. Therefore, present study was designed to demonstrate the effect of Trichosanthes dioica leaves extract against Cisplatin induced renal damage in experimental animals [7].
MATERIALS AND METHODS
Chemicals
All the chemicals used were of analytical grade and procured from Sigma Aldrich Co., USA. Diagnostic kits for urea, Uric acid and BUN were used of Span Diagnostics Ltd. Surat, India.
Animals
Wistar rats (200-250 g) of either sex were procured from Indore college of Pharmacy, Rau Indore, MP, India. They were kept in departmental animal house in well cross ventilated room at 25 ± 1 °C with light and dark cycles of 12 h for 1 week before and during the experiments. All studies were performed in accordance with the guide for the care and use of laboratory animals, as adopted and promulgated by the Institutional Animal Ethical Committee (Reg. No. IIP/CPCSEA/IAEC/2023/03).
Preparation of plant extract
Leaves of T. dioica were collected from local area of Etawah (UP) India, during the month of August. The plant material was identified and the voucher specimen was deposited in the Dept. of Plant Sciences, MJP Rohilkhand University in Bareilly, U.P. (Ref no-3 Dated-5/2/23) Institutional herbarium for future references. The leaves of the Trichosanthes dioica were dried in an oven at 37°C and then powdered with a mechanical grinder, passed through sieve #60 and stored in an air tight container. The dried powdered material (1.0 kg) was refluxed with methanol for three hours. The total filtrate was concentrated to dryness at 40°C and this extract was referred as Trichosanthes dioica leaves extract. The methanol exhausted leaves macerated with water, filtered and concentrated to get aqueous extract.
Nephrotoxicity in rats
Forty-two Wistar rats (200-250 g) were divided into 7 groups of 6 animals each. Group A: control rats that received 0.9% normal saline (i.p.) for 14 days. Group B: Cisplatin-treated rats that received 5 mg/kg (i.p.) for 14 days. Group C: (Cisplatin+ Fosinopril) treated rats that received 5 mg/kg Cisplatin (i.p.) and 5mg/kg Fosinopril (i.p) for 14 days Group D: (Cisplatin + Methanolic Tricosanthes dioica (MTD) 100) treated rats that received 5 mg/kg Cisplatin (i.p.) and 100 mg/kg MTD (p.o.) for 14 days. Group E: (Cisplatin + MTD 200) treated rats that received 5 mg/kg Cisplatin (i.p.) and 200 mg/kg MTD (p.o.) for 14 days. Group F: (Cisplatin + Aqueous Tricosanthes dioica (ATD) 100) treated rats that received 5 mg/kg Cisplatin (i.p.) and 100 mg/kg ATD (p.o.) for 14 days. Group G: (Cisplatin + ATD 200) treated rats that received 5 mg/kg Cisplatin (i.p.) and 200 mg/kg ATD (p.o.) for 14 days After collection of blood and urine, animals were sacrificed by cervical dislocation under mild diethyl ether anaesthesia and kidneys were harvested, rinsed in saline and stored at -80◦C till further biochemical analysis. [8].
Plasma and urine markers of renal damage
Rats of each group were individually housed in metabolic cages for 24 h and urine was collected on the 14th day of the treatment. Blood samples were collected from these overnight fasted animals through retro-orbital plexus puncture in ethylene diamine tetra acetic acid coated vials and plasma was separated by cold centrifugation of vial at 3000 rpm for 10 min. Urea and creatinine were assayed in plasma and urine using commercially available kits (Span Diagnostics Ltd., Surat, India) as per instructions of the manufacturer and blood urea nitrogen (BUN) concentration was also measured as an indicator of renal function [9].
Preparation of renal homogenate
After the completion of the experiment, the kidneys were excised, weighed and homogenized in chilled phosphate buffer (0.1M, pH 7.4) at a concentration of 10% (w/v) (Potter-Elvenhjem glass homogenizer). Homogenates were then centrifuged at 10,000 rpm for 10 min (4 ◦C) at a high speed, supernatant and sediment were used for further biochemical estimations.
Measurement of renal lipid peroxidation
Measurement of malonaldehyde as an index for lipid peroxidation was done using thiobarbituric acid reactive substance assay.
Measurement of renal antioxidants
Superoxide dismutase (SOD) was determined by the ability of the enzyme to inhibition of auto oxidation of pyrogallol. Catalase, GSH and ascorbic acid werer assayed in tissue sediment involve tissue homogenization followed by protein precipitation using sulfosalicylic acid [10,11,12].
Histopathological examination
Pieces of kidney from each group were fixed immediately in 10% neutral formalin for a period of at least 24 h, dehydrated in alcohol and embedded in paraffin, cut into 4-5 µm thick sections and stained with hematoxylin-eosin. The sections were evaluated for the pathological symptoms of nephrotoxicity.
Statistical analysis
The values were represented as mean ± S.E.M. for six rats. Analysis of variance (ANOVA) test was followed by individual comparison by using P. Graph pad Prism software for the determination of level of significance. The values of p<0.05 was considered statistically significant.
RESULTS
Effect of Trichosanthes dioica leaves extract on kidney weight
The effect of various doses of Trichosanthes dioica leaves extract were studied on kidney wt. in Cisplatin induced animals. Renal injury induced by Cisplatin caused significant increase in kidney wt. by 68.00% compared to control group. The percentage protection in kidney wt. of treated groups at 200 mg/kg as 18.46 (P<0.05) Table 1 shows that, Cisplatin treated rats registered significant (p< 0.001) increment in kidney weight.
Effect of Trichosanthes dioica leaves extract on Plasma (Urea, Creatinine, Uric Acid)
Table 1.: Effect of Trichosanthes dioica leaves extract on kidney weight (mg/100 BW), serum urea (mg/dl), serum creatinine (mg/dl), serum uric acid (mg/dl) against Cisplatin induced nephrotoxicity in rats.
|
Group |
Kidney Weight (mg/100 BW) |
Serum |
||
|
Urea (mg/dl) |
Creatinine (mg/dl) |
Urea (mg/dl) |
||
|
Control |
194.12±1.89 |
53.14± 0.30 |
0.69± 23.01 |
1.61±0.35 |
|
Cisplatin |
144.15±1.13 |
74.81 ± 0.25 |
1.94± 0.68 |
2.56 ±0.47 |
|
Standard |
184.45±3.19 |
57. 21± 0.25** |
0.84 ± 0.45** |
1.74 ±0.52** |
|
MTD 100 |
174.87±3.31 |
62.217 ±0.80* |
1.12± 0.48 |
1.94 ±0.16* |
|
MTD200 |
184.21±3.97 |
58.62 ±.45** |
0.91± 0.24* |
1.79 ±0.52** |
|
ATD 100 |
170.92±3.62 |
68.217 ±0.80 |
1.79± 0.48 |
2.19 ±0.22 |
|
ATD 200 |
177.96±4.29 |
63.62 ±.45 |
1.58±0.15 |
2.07± 0.69 |
Values are mean ± S.E.M. of 6 rats in each group
P values: *<0.001 compared with respective control group CONTROL
P values: **<0.05, **<0.01, **<0.001 compared with group (Cisplatin)
Table 2.: Effect of Trichosanthes dioica leaves extract on SOD (Units/mg protein), CAT (µmol of H2O2 consumed/mg protein), GSH (µg/mg protein), and blood urea nitrogen BUN (mg/dl) level against Cisplatin induced nephrotoxicity in rats.
|
Group |
BUN (mg/dl) |
SOD (U/mg protein) |
CAT (U/mg protein) |
GSH (µg/mg protein) |
TBARS (nanomole of MDA/g tissue) |
Urinary Na+ level (mmol/L) |
Urinary K+ level (mmol/L) |
|
Control |
53.14±0.30 |
1.16±0.34 |
0.59±0.19 |
86.23±4.39 |
1.86±0.19 |
109.8 ±5.01 |
4.23 ±0.24 |
|
Cisplatin |
74.81 ±0.25 |
0.81±0.02 |
0.26±0.09 |
35.98±.3.87 |
2.80±0.26 |
137.7± 6.98 |
5.78 ±0.41 |
|
Standard |
57.21±0.25** |
1.12±0.16** |
0.54±0.08** |
84.69±4.62** |
1.95±0.14** |
113.40±2.29** |
4.29±0.08** |
|
MTD100 |
62.217±0.80* |
1.05±0.128 |
0.45±0.02* |
67.49±8.86* |
2.17±0.11* |
127.23± 5.33* |
4.54 ±0.15* |
|
MTD200 |
58.62±.45** |
1.09±0.17** |
0.52±0.02** |
78.33±6.86** |
1.98±0.15** |
118.50±3.95** |
4.31±0.32** |
|
ATD100 |
68.217±0.80 |
0.83±0.05 |
0.32±0.05 |
48.25±7.54 |
2.54±0.12 |
131.50 ±5.12 |
5.08 ±0.13 |
|
ATD200 |
63.62 ±.45 |
0.89±0.02 |
0.39±0.09 |
41.98±.2.64* |
2.31±0.21 |
125.02± 3.81* |
4.97 ±0.78 |
Values are mean ± S.E.M. of 6 rats in each group
P values: *<0.001 compared with respective control group CONTROL
P values: **<0.05, **<0.01, **<0.001 compared with group (Cisplatin)
Effect of Trichosanthes dioica leaves extract on urea and Creatinine in plasma
The effect various doses of Trichosanthes dioica leaves extract were studied on urea and Creatinine in Cisplatin induced animals. Renal injury induced by Cisplatin caused significant changes in renal marker in plasma as urea by 184.81%, Creatinine by 145.64% respectively compared to control group. The percentage protection in renal marker in plasma of treated groups at 200 mg/kg as urea 11.97 (P<0.05), Creatinine 21.85 (P<0.05) (Table 1).
Effect of Trichosanthes dioica leaves extract on urinary Na+ and K+ concentration
The effect various doses of Trichosanthes dioica leaves extract were studied on Na+ and K+ level in Cisplatin induced animals. Renal injury induced by Cisplatin caused significant changed in electrolyte concentration in urine as Na+ by 29.47% and K+ by 56.25% compared to control group. The percentage protection in urine concentration of treated groups at 200 mg/kg as Na+ 17.47 (ns) and K+ 24.25 (P<0.05) were observed (Table 2).
Estimation of renal antioxidants
The percentage changes of SOD, CAT, GSH and ascorbic acid (Table 2) in Cisplatin induced group were as 57.76 (P< 0.001), 67.53 (P< 0.001), 61.5 (P< 0.001) and 32.69 (P< 0.001) respectively. The percentage protection in SOD as 27.13 (P< 0.05), 74.31 (P< 0.001), CAT 51.24 (P< 0.05), 104.1 (P< 0.001) and GSH 32.87 (P< 0.05), 92.37 (P< 0.001) while in ascorbic acid 12.36 (P< 0.05), 21.97 (P< 0.001) at the doses levels 200 mg/kg has shown maximum protection which was almost comparable to those of the normal control.
Histopathological observations
The histological changes in kidneys and pathological manifestations are presented in Figure.1. The nephrotoxicity were confirmed by evaluating the pathological symptoms such as epithelial cells of proximal tubules presented vacuoles and reversible changes, atrophy and inflammatory cell infiltrate, degeneration and desquamation. Treatment with the Trichosanthes dioica leaves extract 200 mg/kg body weight ameliorated the toxic manifestations in the kidney. The histopathological observations supported this conclusion.
Fig 1.: Histological study of kidney tissue in control and experimental groups of rats. (A) No morphological damage was observed in rats in the control group. (B) Histopathological view of renal sections in Cisplatin treated group showed the epithelial cells of proximal tubules presented vacuoles and hydropic changes, atrophy and inflammatory cell infiltration, degeneration and desquamation (indicated by arrows) as compared to control group. (C) Animal treated with ATD-200 mg/kg showed considerably fewer lesions in the tubules and interstitium compared to the group Cisplatin treated rats. (D) Animal treated with MTD-200 mg/kg showed regeneration in tubular epithelial cells.
DISCUSSION
Cisplatin-induced nephrotoxicity is a well-documented event involving some functional and cellular mechanisms such as Glomerular lesions that interfere with Glomerular hemodynamic, and altered tubular transport, with the injury ranging from solely functional lesions to the occurrence of necrosis. Kidney is well recognized to be particularly susceptible to toxic substances. Clinical and investigative toxicity may be clearly described through determining the blood values of blood urea, Serum creatinine, uric acid, sodium and potassium (urine) level for the kidneys. Animal body weight changes brought on the acute toxicity assessments during different stages of the study were not particularly noticeable. Additionally, no changes were found in the mucous membranes, circulation, skin, eyes, or behavior pattern, autonomic nervous system, or central nervous system. Additionally, there were no indications of convulsions, tremor, diarrhea, salivation, sleep, lethargy, or coma. As a result, no outward symptoms of poisoning, toxicity onset, or mortality were seen at this time. When compared to control animals, the nephroprotective activity of Trichosanthes dioica extract in vivo with a single dose of 5 mg/kg i.p. administration of Cisplatin markedly increased the levels of uric acid, blood urea, urine potassium and sodium level, and creatinine in serum, causing an acute renal collapse when biochemically assessed. Plant products may be helpful for renal protection because treatment with Trichosanthes dioica extract at 100 and 200 mg/kg given 16 days prior to the Cisplatin challenge drastically and significantly alters the above values in favor of treatment, establishing the dose at which plant extracts at 100 and 200 mg/kg are necessary for potent protection when challenged by Cisplatin renal toxicity There were no notable effects from the lesser dosage of Trichosanthes dioica aqueous extract (100 mg/kg). According to the current investigation, when confronted with cisplatin toxicity, methanol extracts (200 mg/kg) have a strong and noteworthy nephrotoxicity prevention capability. Histopathological results demonstrating structural changes in renal tissue of aminoglycoside antibiotics such as Cisplatin were reported by some researchers [32]. Histopathological view of renal sections in Cisplatin treated group showed the epithelial cells of proximal tubules presented vacuoles and hydropic changes, atrophy and inflammatory cell infiltrate, degeneration and desquamation. Vacuoles and hydropic changes, atrophy and inflammatory cell infiltrate, degeneration and desquamation and epithelial changes were considerably mild in the groups treated with Cisplatin + MTD200 mg/kg showed regeneration in tubular epithelial cells. We think that, morphological changes in kidneys were because of Cisplatin injection, but these changes tended to be considerably mild in Cisplatin + Trichosanthes dioica leaves extract treatment.
CONCLUSION
Mudit Kumar, Pushpendra Kumar, Mona Piplani*, Nephroprotective Potential of Trichosanthes Dioica Roxb. Leaves Extract Against Cisplatin Induced Nephrotoxicity in Albino Rats, Int. J. Med. Pharm. Sci., 2026, 2 (4), 59-64. https://doi.org/10.5281/zenodo.19474122
10.5281/zenodo.19474122