INTRODUCTION:

Functioning of the urinary system is depending on the adequate functioning of kidneys which is essential for the management of internal stability in an organism (homeostasis), acid-base balance and regulation of blood pressure¹. The most common kidney problem is nephrotoxicity, which occurs when the body is exposed to drugs or toxins.

Various synthetic chemicals and pharmacologically active agents such as aminoglycosides antibiotics, NSAIDs and other drugs can adversely affect the kidney like acute renal failure, chronic interstitial nephritis, and nephritic syndrome can occur. kidneys are incapable to excrete out metabolic by-products like BUN, urea, creatinine etc. Exposure to chemical reagents like ethylene glycol, carbon tetrachloride, sodium oxalate and heavy metals such as lead, mercury, cadmium, and arsenic also induces nephrotoxicity. Prompt recognition of the disease and cessation of responsible drugs are usually the only necessary therapies^2,3.

An Aminoglycoside like Gentamicin is a broad-spectrum antibiotic in combination with beta-lactams for the treatment of systemic infection⁴. The major risk associated with Gentamicin is nephrotoxicity and ototoxicity. Around 10-25% of patients are susceptible to developing Acute renal failure at the therapeutic dose levels. AKI can be estimated in terms of an increase in nitrogenous waste products in the blood like blood urea nitrogen [BUN], serum total proteins and serum creatinine, kidney levels of glutathione (GSH). It is also observed by various morphological and functional alterations the in the renal tubules leading to tubular necrosis^5,6. Medicinal plants have curative properties due to the presence of various complex chemical substances. In the Indian traditional system of medicine, several medicinal plants are prescribed for alleviating renal damage and treating kidney complications and early literature has prescribed various herbs for the cure of renal disorders^7,8.

In the present study, the hydroalcoholic extracts of four Indian medicinal plants were selected to investigate and compare the nephroprotective potential. The nephroprotective potential of Hydroalcoholic extracts of stem bark of Ficus religiosa, stem of Tinospora cordifolia, leaves of Moringa oleifera and roots of Boerhaevia diffusa have not been compared yet. Different parts of all these selected plants showed profound nephroprotective and curative effects in different drug-induced nephrotoxic animal models^9,12.

Ficus religiosa Linn. (Moraceae) is called “Pipal tree” in Hindi. It has medicinal value in the Indian traditional and folk medicine system, and it is useful in the treatment of ulcers^13,14, hepatic disorders, kidney disorders, different microbial infections, diabetes and neurodegeneration^15,22.

Tinospora cordifolia (Menispermaceae) called “Giloy” in Hindi, is a perennial climber shrub¹⁷. In the ayurvedic medicine system, it is used for the treatment of diseases like jaundice, weakness, blood disorders, fever, viral hepatitis, inflammation, microbial diseases, dyspepsia, urinary diseases, skin diseases, allergic condition, diabetes and hepatoprotective^23,27 etc.

Moringa oleifera (Moringaceae), is widely distributed in different countries²⁸. Almost every part of the plant has been used for various pharmacological activities such as analgesic, antifungal, antibacterial, antihypertensive and antispasmodic, antioxidant properties, anti-hyperlipidaemic and anti-atherosclerotic, anticancer, anti-anxiety, antipyretic, free radical scavenging agent and wound healing^29,36.

Boerhaevia diffusa Linn (Nyctaginaceae), is known as Punarnava in Sanskrit and Hogweed in English. It is native to India and is a perennial creeping weed or ascending herb³⁷. It rejuvenates the liver, male reproductive system and cleanses the kidneys and helps to get rid of renal calculi, immunomodulator, hepatoprotective, antidiabetic, antibacterial, antistress, antimalarial^38,43.

MATERIALS AND METHODS:

Plant material:

In the present study, all the selected parts - such as stem bark of Ficus religiosa, stem of Tinospora cordifolia, root of Boerhaevia diffusa and leaves of Moringa oleifera were collected and authenticated by Dr Md. Mustafa, Botanist, Department of Botany, Kakatiya University, Telangana (Accession no. 1092, 1094, 1096, 1098).

Preparation of extracts:

Dried parts of selected plants were powdered by using a mechanical grinder. The powders were passed through mesh sieve 44 and stored in airtight containers. The powdered sample was defatted with petroleum ether and kept for 72 hours at room temperature. 100 gm of each dried powdered sample was extracted with a mixture of ethanol and distilled water (70:30) by using the Soxhlet Apparatus for 24 hours. The solvent was removed to get the solid extract. The percentage yield of extracts for Ficus religiosa (4.75%), Tinospora cordifolia (5.67%), Boerhaevia diffusa (4.62%) and Moringa oleifera (7.54%).

Sub-Acute toxicity studies:

The acute oral toxicity was carried out as per the OECD guidelines no. 425. For acute toxicity studies, 3 animals of a single sex had been selected for each extract and the dose 5, 50, 300 and 2000mg/kg body weight was selected. Hydroalcoholic extracts of all the selected parts of Indian medicinal plants were found to be safe to dose levels of 2000mg/kg per oral. The LD₅₀calculation was done as per Karber’s method and 1/10 dose was selected for animal study⁴⁴.

Experimental study design:

A total of Sixty-six (66) adult albino Wistar rats of either sex were inducted into the study following simple random sampling. The study proceeded on adult Wistar rats weighing in the range of 140 - 160g. Animals were kept under standard conditions the temperature was maintained at 27±4.5°C and relative humidity is 60% with periodic 12 hours of daylight and 12 hrs dark cycle. Animals were confined into hygienic cages and fed with standard diet pellets with water ad libitum. The experimental protocol proceeds as per the CPCSEA norms (IAEC Approval No: CPCSEA/ IAEC/JLS/ 16/ 07/21/0470.

The albino Wistar rats were divided into Eleven groups each group containing six rats and treated with hydroalcoholic extract of Ficus religiosa, Tinospora cordifolia, Moringa oleifera and Boerhaevia diffusa for 14 days as per animal activity protocol given in Table 1.

Table 1. Grouping of animals

Group	Group Name	Treatment
Group- I	Normal	Received Normal water
Group-II	Standard	Received silymarin in a single dose of 100 mg/kg/p.o daily
Group-III	Negative control	Received gentamicin in a single dose of 100 mg/kg/ i.p. daily
Group-IV	FRHL	Received 200 mg/ kg/p.o of Hydroalcoholic extract of Ficus religiosa with gentamicin 100 mg/ kg/ i.p. /per day
Group-V	FRHH	Received 400 mg/kg/p.o of Hydroalcoholic extract of Ficus religiosa with gentamicin 100 mg/kg/ i.p. /per day
Group-VI	TCHL	Received 200 mg/ kg/p.o of Hydroalcoholic extract of Tinospora cordifolia with gentamicin 100 mg/kg/ i.p. /per day
Group-VII	TCHH	Received 400 mg/ kg/p.o of Hydroalcoholic extract of Tinospora cordifolia with gentamicin 100 mg/ kg/ i.p. /per day
Group-VIII	MOHL	Received 200 mg/ kg/p.o of Hydroalcoholic extract of Moringa oleifera with gentamicin 100 mg/ kg/ i.p. /per day
Group-IX	MOHH	Received 400 mg/ kg/p.o of Hydroalcoholic extract of Moringa oleifera with gentamicin 100 mg/ kg/ i.p. /per day
Group-X	BDHL	Received 200 mg/ kg/p.o of Hydroalcoholic extract of Boerhaevia diffusa with gentamicin 100 mg/ kg/ i.p. /per day
Group-XI	BDHH	Received 400 mg/ kg/p.o of Hydroalcoholic extract of Boerhaevia diffusa with gentamicin 100 mg/ kg/ i.p. /per day

Where p.o - Per Oral, i.p. - Intraperitoneal

Estimation of Biochemical parameters:

After the completion of the treatment protocol, parameters change in body weight was checked and 24 hours urine was collected for assessment of different urine parameters. Then all the animals had been anaesthetized with ketamine intraperitoneal injection and blood have been collected in tubes from the retro-orbital venous plexus biochemical assays. The collected blood samples have been centrifuged and separated serum was used for the estimation of serum parameters like BUN and serum creatinine, creatinine clearance and total protein^45,50.

Estimations of oxidative stress biomarkers in kidney tissue homogeneity:

For the study, kidneys were washed with ice-cold water and homogenized in phosphate buffer (0.1 M, pH 7) at 10% (w/v). A small portion of this is utilized for the assessment of malondialdehyde (MDA) as a marker for lipid peroxidation. Assessment of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) was done with the supernatant got by centrifugation of the remaining portion of homogenate^51,53.

Histopathology of Kidney:

After the collection of blood samples, all the animals were sacrificed and without delay, kidneys had been eliminated and glued in 10% formalin for histopathological assessment. Then it is implanted in paraffin and sections were stained with hematoxylin and eosin (H&E).

Statistical analysis:

Statistics are communicated as Mean ± SEM and statistical analysis was analyzed by using Two Way ANOVA followed by multiple comparison tests. *p<0.01 and **p<0.05 compared to standard animals. GraphPad Prism V five. 02 became applied for measurable evaluation and charting.

RESULT AND DISCUSSION:

In this study, the nephroprotective effect of hydroalcoholic extract of stem bark of Ficus religiosa, stem of Tinospora cordifolia, leaves of Moringa oleifera and roots of Boerhaevia diffusa used at two dose levels (200mg/kg and 400mg/kg) was estimated in Gentamicin induced nephrotoxic Wistar rats.

The animals showed severe body weight loss was observed in Gentamicin treated animals when compared with the standard group. Pre-treatment with Tinospora cordifolia and Boerhaevia diffusa extract significantly (183.33±3.18, 179.66±3.26) attenuated the Gentamicin-induced decrease in body weight in comparison to other plant extract treatment animals⁵⁴.

As shown in Table 2, Silymarin and Plant extract (TCHH and BDHH) produced a marked increase in urine volume of 4.5±0.83ml/180 min and 4.16±0.40ml/180 min and urine creatinine. On the contrary, Gentamicin induced a severe reduction in diuresis and urine creatinine, as expected. The effect on urine parameters by TCHH and BDHH was characterized by a dose-dependent (p < 0.05) in the urine output in normal rats.

Table 2: Effect of Hydroalcoholic extracts on body weights and urine parameters in Gentamicin Induced Nephrotoxic Wistar Rats

S. No	Group	Initial body weights (g)	Final body weights (g)	Urine volume (ml)	Urine creatinine (mg/dl)
1	Normal	150±3.16	179.5±3.39	4.5±0.83	3.48±0.12
2	Standard	148.33±2.58	190.33±4.54**	4.83±0.75	7.78±0.23
3	Negative control	152.5±5.24	129.66±3.26*	2.33±0.51**	4.66±0.51
4	FRHL	150.83±3.76	148±2.44	2.15±0.28	5.33±0.51
5	FRHH	150±4.47	152.5±2.73	3.15±0.62	6.33±0.81*
6	TCHL	149.16±4.90	156.33±2.16	2.66±0.51*	6.83±0.75*
7	TCHH	150.83±4.91*	183.33±3.18**	4.5±0.83**	8.33±0.51
8	MOHL	151.66±4.08	152.83±2.48	2.5±0.83	5.33±0.51
9	MOHH	150.83±3.89	163.5±1.97	3.66±1.21	6.83±0.75
10	BDHL	150.38±4.91	157.16±2.48	2.83±0.42*	6.33±0.51*
11	BDHH	152.5±2.73	179.66±3.26**	4.16±0.40**	7.16±0.75

n=6, Values are expressed as Mean ± SEM and statistical analysis was carried out by One Way ANOVA followed by Dunnett’s multiple comparison test. *p<0.01 and **p<0.05 compared to standard animals.

Table 3: Effect of Hydroalcoholic extracts on different biochemical parameters in Gentamicin Induced Nephrotoxic Wistar Rats

S. No	Group	BUN (mg/dl)	Serum creatinine (mg/dl)	Total protein (gm/dl)	Creatinine clearance (ml/min)
1	Normal	13.16±1.60	0.18±0.01	5.36±0.25	1.4±0.12
2	Standard	11±0.89	0.15±0.01	5.15±0.10	1.45±0.07
3	Negative control	50.16±1.83	1.33±0.02	16.86±0.76	0.15±0.08
4	FRHL	37.16±1.83	1.31±0.02	16.16±0.75	0.19±0.09
5	FRHH	26.83±1.16*	1.03±0.05	12.33±0.51	0.28±0.02*
6	TCHL	28.66±1.62*	1.03±0.09	11.66±0.81*	0.78±0.01*
7	TCHH	16.83±2.13**	0.48±0.40**	7.33±0.81**	1.4±0.08**
8	MOHL	32.16±2.03	1.23±0.04	12.5±2.07	0.37±0.02*
9	MOHH	22.5±1.64*	0.75±0.05*	8.33±0.81*	0.78±0.01
10	BDHL	32.66±2.33	1.11±0.07	11.16±0.75	0.76±0.02*
11	BDHH	18.33±1.50**	0.52±0.07*	7.33±1.03*	1.22±0.03

n=6, Values are expressed as Mean ± SEM and statistical analysis was carried out by One Way ANOVA followed by Dunnett’s multiple comparison test. *p<0.01 and **p<0.05 compared to standard animals.

Table 4: Effect of Hydroalcoholic extracts on oxidative stress biomarkers in Gentamicin Induced Nephrotoxic Wistar Rats

S. No	Group	SOD (U/mg Tissue)	CATALASE (µmoles of H₂O₂consumed/min/m g protein)	MDA (moles /mg protein)
1	Normal	17.5±0.52	195.83±0.78	2.33±0.89
2	Standard	15±1.67	190±2.89	2.16±0.40
3	Negative control	6.33±0.51	151.33±3.20*	4.16±0.40*
4	FRHL	7.50±0.54	158.83±2.40*	3.16±0.40
5	FRHH	9.33±0.51	168.33±1.50	2.5±0.54
6	TCHL	10.05±0.54*	184.33±2.65	3.16±0.40
7	TCHH	12.66±0.51**	191.16±0.75	2.16±0.40**
8	MOHL	9.16±0.40	161.5±3.01*	3.16±0.40
9	MOHH	10±0.63*	179.16±1.60**	2.33±0.51**
10	BDHL	9.33±0.51	170.33±2.33	3.66±0.51
11	BDHH	12.66±1.03**	185.16.16±1.94	2.16±0.40

n=6, Values are expressed as Mean ± SEM and statistical analysis was carried out by One Way ANOVA followed by Dunnett’s multiple comparison test. *p<0.01 and **p<0.05 compared to standard animals

Estimation of Biochemical parameters:

In the present study, Gentamicin-induced nephrotoxicity was marked by significantly increased levels of BUN, serum creatinine, total protein and reduced levels of creatinine clearance when compared to the control group (p<0.05). As shown in Table 3, Silymarin and Plant extract TCHH (16.83±2.13) and BDHH (18.33±1.50) produced a marked decrease in elevated levels of blood urea nitrogen (BUN), serum creatinine, and total protein when compared to the standard control group (II) (p<0.05). Creatinine clearance was significantly decreased in the negative group^55,56. In TCHH and BDHH-treated groups, creatinine clearance was restored to standard and normal groups. The results are shown in Table 3.

Estimations of oxidative stress biomarkers:

There was a significant decrease in the SOD and catalase levels and an increase in MDA levels in Gentamicin treated negative control group when compared to the normal group (group I) indicating the excessive production of free radicals due to the oxidative stress caused by Gentamicin (p<0.05)^57,59. Silymarin and plant extract TCHH and BDHH normalized the superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) levels indicating the antioxidant properties of the plant extract selected in the present study (p<0.05). The significance in the plant extract-treated groups was dose-dependent. The results are shown in Table 4

Histopathology of kidney:

Kidneys of the normal group showing cortical and medullary structure Renal tubules and the renal corpuscle’s structure show regularly. The cortex confirmed renal corpuscles organized in parallel rows at the proper attitude to the fibrous capsule constituting the cortical labyrinths. The cortical labyrinths alternated regularly with the medullary rays (Fig 7a). Kidneys of the standard group revealed a normal renal cortical architecture with alternating areas of the cortical labyrinth and medullary rays. The tubular lumen was not dilated as compared with the negative group animals. PCTs cells are looking normal (Fig 7b). The kidney of the negative control group revealed irregular cortical labyrinth and medullary rays. The PCT’s cell lining also looks slightly enlarged and swollen as compared because of vacuolization in their cytoplasm. The nuclei in many of the cells were enlarged and displaced from their central or basal locations (Fig 7c). The kidneys of FRHL treated showed tubular dilatation and vacuolar degeneration of the PCT cells. Broken basement membrane and irregular epithelial debris in the lumen (Fig 7d). The kidneys of FRHH treated rat’s kidney showed disrupted basement membranes in some of the proximal tubules. The nuclei are condensed, and epithelial and nuclear debris is seen (Fig 7e). The kidneys of TCHL treated rat’s kidney showing mild glomerular injury, distorted glomeruli architecture, minimal vacuolation and normal vascularity (Fig 7f). The kidneys of TCHH treated rats showed almost normal PCT and DCT with intact cells and nuclei, comparable to the Negative group (Fig 7g). The kidneys of MOHL treated rats showed enlarged vacuolar degeneration with edema of both the proximal and distal convoluted tubular epithelium (Fig 7h). The kidneys of MOHH treated rats showed mild tubular dilatation, vacuolar degeneration of the PCT cells, and no presence of epithelial debris in the lumen (Fig 7i). The BDHL treated rat’s kidneys show gross glomerular edema, increased vascularity, interstitial edema and disturbed tissue architecture (Fig 7j). In the kidneys of BDHH treated rat’s kidney with slight glomerular edema and distortion, glomerular details are visible, such as hyperemia and mild interstitial edema (Fig 7k).

CONCLUSION:

Gentamicin is a very commonly used aminoglycoside and nephrotoxicity is a common side effect of all aminoglycosides. Due to this, it has restricted use, within the current look, gentamicin infusion for multi-week activates a big markdown in the outline weight of rodents. This weight change may result from direct damage in renal tubules following insufficiency of the rounded cells to reabsorb water, essential to parchedness and absence of facet weight or similarly evolved catabolism resulting in acidosis, anorexia, and diminished consumption.

This study empowers to discover nephroprotective plant through the assessment of hydroalcoholic extract of stem bark of Ficus religiosa, stem of Tinospora cordifolia, leaves of Moringa oleifera and underlying foundations of Boerhaevia diffusa utilized at doses of 200, 400 mg/kg to reverse the gentamicin induced nephrotoxicity. Results of urine parameters, blood serum parameters and histopathological slides of the kidney showed that hydroalcoholic extract of stem of Tinospora cordifolia high dose (TCHH) and roots of Boerhaevia diffusa high dose (BDHH) reversed the changes of Gentamicin-induced nephrotoxicity in Wistar rats at a dose level of 400 mg/kg. Histopathological study of kidneys confirmed the results of biochemical parameters for TCHH and BDHH. Other extracts also showed dose-dependent nephroprotective effects. In the end, TCHH and BDHH address a shiny new solution for Gentamicin-induced nephrotoxicity; be that as it could, a more noteworthy examination is needed to choose its clinical aspect.

In the end, TCHH and BDHH address a shiny new solution for Gentamicin-induced nephrotoxicity; be that as it could, a more noteworthy examination is needed to choose its clinical aspect. Further, the study can be extended by making herbal formulations with the use of these extracts.

ACKNOWLEDGEMENT:

The authors acknowledge the recognition of Jeeva Life Sciences to provide the facility to carry out this work. We also want to support our faculty who provide continuous support during the study.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest

List of Abbreviations:

S. No.	Plants	Abbreviations for Hydroalcoholic extract (Low dose) 200mg/kg	Abbreviations for Hydroalcoholic extract (high dose) 400mg/kg
1	Ficus religiosa	FRHL	FRHH
2	Tinospora cordifolia	TCHL	TCHH
3	Moringa oleifera	MOHL	MOHH
4	Boerhaevia diffusa	BDHL	BDHH

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Received on 14.07.2022 Modified on 16.10.2022

Research J. Pharm. and Tech 2023; 16(9):4307-4313.

DOI: 10.52711/0974-360X.2023.00705