INTRODUCTION:

Aminoglycosides have historically ranked among the most frequent culprits in drug-induced kidney damage.¹Chronic renal failure is a major ailment that impacts a staggering 10% of the world's population, totaling over 800 million people. This ailment tends to be more prevalent among older adults, women, and those dealing with diabetes mellitus and high blood pressure.^2,3 Recent literature is shedding light on the fundamental mechanism behind gentamicin-induced kidney damage.

Systematic research has proposed that gentamicin (GM) elevates the generation of redox radicals and oxidative stress molecules within the kidney. ROS serve as catalysts for oxidative stress, acting as a promotor to activate inflammatory signaling cascades and trigger programmed cell death.^4,5

Cansjera rheedii, commonly known as Kalimanakeerai in Tamil and belonging to the Opiliaceae family, is a climbing shrub primarily found in India. This plant exhibits several noteworthy properties, including anti-inflammatory activity⁶, diuretic activity⁷, anti-diabetic activity, anti-oxidant activity, anti-microbial activity, anti-helminthic activity⁸, and anti-pyretic activity.⁹ Its chemical composition comprises flavonoids, saponins, glycosides, phenolic compounds, alkaloids, and tannins.¹⁰ In traditional practices, a leaf of this plant is used to treat kidney disorders.¹¹ The precise mechanism underlying the nephroprotective potential of Cansjera rheedii remains unexplored. Drawing inspiration from existing literature, we designed an experimental study to assess the renal defense activity of Cansjera rheedii on gentamicin-instigated renal toxicity in Wister albino rats and to investigate potential mechanisms for reversing kidney damage.

METHODOLOGY:

Herbal plant material:

The fresh leaves of the Cansjera rheedii were gathered in January 2022 from the Agumbe forest in Shimoga district, Karnataka. Authenticated by Shri M.K Ganachari HOD Botany department Basaveshwar Science College, Bagalkot, Karnataka. A plant specimen labeled as (BSc/Bot/2022/05) was then deposited in the college's herbarium for future reference.

Methods:

Preparation of plant leaf extract by cold maceration:

The fresh leaves of Cansjera rheedii were meticulously cleaned and shade-dried for 2 weeks. These dry leaves were then finely grounded. Subsequently, the fine powder of leaf was subjected to a three-day maceration process with a hydro-alcoholic solution at room temperature. After the maceration period, the solvent underwent distillation further allowing it to naturally evaporate at room temperature, leading to the formation of a concentrated extract. Subsequently, the evaporated extract stored at 2-4^°C.

Studies on acute oral toxicity:

OECD guideline number 425 is followed for acute oral toxicity studies of leaves extract HECR.¹²

Qualitative phytochemical screening of hydro-alcohol extract of Cansjera rheedii:

Qualitative chemical investigations were conducted on the plant extract to identify phytoconstituents, including carbohydrates, alkaloids, tannins, saponins, glycosides, flavonoids, proteins and phenolic compounds. To perform phytochemical tests, a 1-gram sample of Cansjera rheedii hydro-alcohol extract was dissolved in 100ml of its mother solvent. This resulted in a concentration of 1% (w/v). The well-established procedures were utilized for identifying the various phytochemical compounds present in plant extract obtained. The name "HECR" (Hydro-alcohol extract of Cansjera rheedii) was assigned to the extract.^13,14,15

HPLC methodology to examine the quercetin content within the hydroalcoholic extract of Cansjera rheedii

Preparation of Standard and Sample Solutions:

An accurate measurement of 1mg quercetin is taken. Next, the substance was poured into a flask with a volume of 10ml and filled with methanol. The process commenced by diluting the solution with methanol, yielding a stock solution at a concentration of 20µg/ml. The extract from Cansjera rheedii, weighing 100mg, was added to a 10ml volumetric flask. It was then dissolved in methanol, producing a solution with a concentration of one milligram per milliter. A 0.45µm-diameter nylon syringe filter was also used to filter the mixture.¹⁵

Mobile Phase:

When choosing the phase for quercetin, we took into account factors such as solubility, stability, and suitability. The most effective phase we discovered was a combination of methanol and orthophosphoric acid (0.5% concentration) at a volumetric ratio of 80:20%. Quercetin detection at a wavelength of 400nm was made possible through the use of this technique.¹⁶

Chromatographic Conditions:

Table no: 1 chromatographic condition of Quercetin

Chromatographic Condition	Quercetin
Operation system	Gradient elution
Constant ambient temperature	35 degrees Celsius
Moving phase	Methanol : 0.5% Orthophosphoric acid (80%: 20% v/v)
Fixed phase	Enable C18G (4.8 X 250 mm, 5 µ particle size)
Wavelength (λ)	400nm
Flow speed	1ml/minute
Running time	10min
Sample injected	10µl

Sample Analysis:

The HPLC chromatograms of the hydroalcoholic extract of Cansjera rheedi were developed using the same conditions as the standard quercetin. The quercetin calibration curves were used to determine the corresponding concentration of quercetin based on the respective peak area values.

Experimental animals:

The Wistar albino rats weighing (220 – 250g) from the Animal House of BVVS Hanagal Shri Kumareshwar College of Pharmacy Bagalkote. The animals will undergo an acclimation period to adjust to the laboratory conditions at room temperature before the experimentation begins. The animals were provided with basic facilities, such as access to food and water, and were kept in pairs in cozy plastic cages with soft bedding. The permission to carry out research work given by the IAEC (Ref. No: IAEC/HSKCOP/AUG2022/PG5).

Animals grouping for gentamicin-induced renal toxicity in Wistar albino rat:

Group 1 : The Normal group receives normal saline

Group 2 : The Control group receives gentamicin for 8 days

Group 3: The Standard group receives gentamicin+ (selenium 2mg/kg, p.o)

Group 4: The Treatment group receives gentamicin+ (HECR 200mg/kg, p,o)

Group 5: The Treatment group receives gentamicin+ (HECR 400mg/kg, p,o)

Each group consists of six animals. Group (2-5) induced gentamicin (100mg/kg) i.p along with treatment for 8 days.

Biochemical Estimation:

Analysis of serum sample:

Blood samples, totaling two milliliters, were procured by puncturing the retroorbital venous plexus of every animal. These samples were then transferred to centrifuge tubes, devoid of any anticoagulant. Afterward, the samples were permitted to coagulate at ambient conditions. Afterward, the serum sample was separated by placing them in a cold research centrifuge machine and centrifuging at 1500rpm for 20min. For the evaluation of serum BUN, creatinine and uric acid. The estimations were carried out by standard available kits. It was analyzed using the UV-Spectrophotometer 1900i by Shimadzu Japan.^17,18

Collection and estimation of urine samples:

On 8^th day rats were placed in separate cages and 24hrs urine collected. The urine samples were estimated for creatinine, BUN and uric acid. Analysed by reagent kits.¹⁹

Evaluation of renal homogenate antioxidant parameters:

On the eighth day, the rats were euthanized kidneys removed and cleaned in chilled 0.15M potassium chloride solution. To finely mince the right kidney, scissors were used before homogenizing it with cold buffer phosphate (0.1 M, pH 7). The resultant kidney tissue sample was then centrifuged at 10,000rpm for 10 minutes at 4°C in a chilled laboratory centrifuge. The determination of catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and lipid peroxidase (LPO) levels was carried out using the kidney homogenate.^20,21,22,23

Histopathological studies:

First, the kidneys that have been isolated will be weighed and then placed into a solution of 10% formalin with a pH of 7. In the paraffin-fixed kidney section, the pathological alterations can be identified upon staining with hematoxylin and eosin.

Statical analysis:

All values are expressed in Mean±SEM [n = 6] first One-way analysis of variance test [ANOVA] carried out further Dunnette’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, superscripts represent comparison to control group. # = ***p<0.001, as compared to the normal group.

RESULTS:

Acute oral toxicity:

The animals displayed regular breathing patterns, with no signs of tremors, convulsions, salivation, or diarrhea observed. Observations conducted over 14 days found no evidence of toxicity in animals. The safety doses were subsequently determined to be 200mg/kg and 400 mg/kg.

Qualitative Phytochemical screening of hydro alcoholic extract of Cansjera rheedii:

A preliminary screening of the plant extract showed the existence of carbohydrates, glycosides, alkaloids, phytosterols, saponins, flavonoid, proteins, amino acids, tannin and phenolic compounds represented in Table 2

Table 2: Qualitative phytochemical test of hydro-alcoholic extract of Cansjera rheedii

Active compounds	Type of test	Result
Carbohydrates	Molish’s reagent	+
Carbohydrates	Fehling’s reagent	+
Glycosides	Legal’s test	+
	Baljet’s test	-
	Bontrager’s test	+
Alkaloids	Mayer’s reagent	-
	Dragendorff's reagent	+
	Hager’s reagent	-
	Wagner’s reagent	-
Saponins	Foam test	+
Phytosterols	Salkowski test	+
Phytosterols	Liebermann Burchard test	+
Flavonoids	Shinoda test	-
	FeCl₃ reagent	+
	Alkaline reagent	-
	Lead Acetate solution	+
Tannins and Phenolic Compounds	5% FeCl₃ solution	+
	Lead acetate solution	+
	^{Dilute HNO}3	+
Proteins and Amino acids	Biuret reagent	-
	Million’s reagent	+
	Ninhydrin reagent	-

Positive (+), Negative (-)

Quantification of the amount of quercetin present in the hydroalcoholic extract of Cansjera rheedii using HPLC analysis:

HPLC analysis of chromatogram of quercetin at 400 nm

(Fig-1A) Standard quercetin HPLC chromatogram at 400 nm

(Fig-1B) HPLC chromatogram of HECR

Quercetin content in hydroalcoholic extract of Cansjera rheedii:

The standardization of extract was confirmed by the result of the quercetin content in the hydroalcoholic Extract of Cansjera rheedi which was 0.1220±0.0075% w/w quercetin. The results are represented in figure (1A,1B).

Effect of hydroalcoholic extract of Cansjera rheedii (HECR) on serum, urine, and antioxidants parameters on gentamicin-induced renal toxicity in rats:

Renal biomarkers such as creatinine, BUN, uric acid in urine sample and blood serum in all groups of animals (Gr-1 to Gr-5) were measured and represented in Table no 3 and Table no 4. Antioxidant parameters of all the rats (Gr-1 to Gr-5) were measured and given in Table 5. Upon induction of gentamicin 100mg/kg i.p for eight days to rats caused a rise (p<0.001) in levels of renal biomarkers creatinine, BUN and uric acid in both serum and urine as comparison with the normal group (Gr-I). Standard drug selenium caused a decrease in serum and urine creatinine, BUN and uric acid as compared to control (Gr-II) respectively. HECR low dose 200mg/kg and 400mg/kg exhibited a marked decrease in serum and urine creatinine, BUN, and uric acid as compared to control (Gr-II). gentamicin caused a significantly reduced in the levels of SOD, CAT and GSH in (Gr-II) control when compared to the normal (Gr-I) of rats. selenium treatment elevated the levels of antioxidant enzymes and decreased level of LPO with comparison to the control (Gr-II). Treatment with HECR 200mg/kg & HECR 400mg/kg significantly increased the levels of SOD, GSH, CAT and decreased level of LPO as compared to the control (Gr-II).

Table 3: Effect of hydro-alcoholic extract of Cansjera rheedii (HECR) on blood serum Creatinine, BUN, and Uric acid against gentamicin induced renal toxicity in Wistar rat

Groups

Creatinine (mg/dl)

BUN (mg/dl)

Uric acid (mg/dl)

Normal

0.6034±0.05821

21.98±3.110

4.311±0.3259

Control

6.089±

0.5172^#

319.8±

71.94^#

19.58±

0.5823^#

Standard

0.8655±

0.05284***

116.3±

24.40 **

7.853±

0.4731 ***

HECR

(200mg/kg)

3.089±

0.8143***

145.8±

45.42 *

12.32±

1.512 ***

HECR (400mg/kg)

1.289±

0.1176***

105.1±

16.44 **

11.89±

1.359 ***

Table 4: Effect of hydro-alcoholic extract of Cansjera rheedii (HECR) against urine Creatinine, Uric acid and BUN gentamicin induced renal toxicity in Wistar rat

Groups	Creatinine (mg/dl)	BUN (mg/dl)	Uric acid(mg/dl)
Normal	0.7069±0.06343	21.69±5.433	4.395±0.3890
Control	10.16±1.343^#	192.6±20.84^#	11.47±1.812^#
Standard	3.655± 0.4619***	81.54± 7.900***	5.526± 0.2118***
HECR (200mg/kg)	8.378± 0.3762*	142.7± 11.69*	9.316± 0.1955**
HECR (400mg/kg)	5.910± 0.6273**	110.2± 10.68***	6.868± 0.6140**

Table 5: Effect of hydro-alcoholic extract of Cansjera rheedii (HECR) on Lipid peroxidation and antioxidant enzymes in renal homogenate against gentamicin induced renal toxicity in Wistar rat

Groups

LPO

SOD

GSH

CAT

Normal

83.86±

30.94

613.0±

36.73

33.70±

4.841

0.4929±

0.02506

Control

541.0±

146.2^#

172.0±

29.89^#

11.83±

0.7175^#

0.1040±

0.01889^#

Standard

116.2±

10.44 **

574.1±

53.68***

26.92±

1.405**

0.4158±

0.05321***

HECR (200mg/kg)

383.8±

50.82*

429.9±

28.39***

19.74±

2.557*

0.2872±

0.03902*

HECR (400mg/kg)

194.7±

10.55 **

470.9±

48.75***

24.36±

1.809**

0.3395±

0.06005**

LPO,SOD&CAT - (Units/mg of protein), GSH-(µMol/mg protein)

All values are expressed in Mean ± SEM [n = 6] first One-way analysis of variance test (ANOVA) carried out further Dunnette’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, superscripts represent comparison to control group. # = ***p<0.001, as compared to the normal group.

Histopathological Examination of the kidney section: Hematoxylin and eosin stain 40x.

(Figure-2)

Normal group:

In normal group (1) the proximal tubules and the glomerulus (G) exhibit a typical structural appearance.

Control group:

In the disease control group (2) the kidneys display greater mononuclear cell infiltration, areas of necrosis heightened damage to the glomerulus (G), and nearly complete desquamation in the proximal renal tubules.

Standard group:

In the standard group (3) The tubular epithelial cells show mild desquamation and atrophy, accompanied by minor alterations in the glomerulus (G)

Treatment group 200mg/kg:

In the treatment group (4), administration of HECR at 200mg/kg resulted in mild to moderate damage to the glomerulus (G), an insignificant desquamation space, and necrosis of the renal tubules.

Treatment group 400mg/kg:

In the treatment group (5) receiving HECR at 400mg/kg, there was a restoration of architecture, reduced damage to the glomerulus (G), a decreased desquamation space, and diminished necrosis of the renal tubules.

DISCUSSION:

Gentamicin, also known as GM, it is a class of aminoglycosides for treating dangerous bacterial infection with bacteria with negative Gram stain organisms. However, a significant drawback associated with its usage is the potential for inducing both ototoxicity and nephrotoxicity.^24,25 The generation of free radicals is enhanced in cases of nephrotoxicity induced by gentamicin, potentially due to the inactivation of antioxidants such as Superoxide-neutralizing enzyme and cellular defense molecule GSH. Numerous studies have confirmed a clear association between nephrotoxicity and oxidative stress. This kidney dysfunction is evidenced by elevated levels of serum creatinine and urea, as well as increased kidney tissue malondialdehyde (MDA) levels, indicative of lipid peroxidation. Additionally, MDA holds significant importance due to its essential function in upholding cell integrity and involvement in cell metabolism.^26,27 The hydro-alcoholic extract of Cansjera rheedii was analyzed in this study to determine its qualitative phytochemical composition. Giving gentamicin at a dosage of 100mg/kg for a period of 8 days in rats resulted in notable increases in renal biomarkers including creatinine, BUN and uric acid levels serum and urine within the disease control group. The oral administration of Cansjera rheedi leaves extract (HECR) at both doses resulted a considerable reduction in renal biomarkers compared to the disease control group. The herbal extract treatment group experienced a noteworthy reduction in MDA levels, while antioxidants such as SOD, CAT, and GSH exhibited a marked increase as compared to the disease animals. These findings strongly suggest the presence of antioxidants within the Cansjera rheedi (HECR) extract. In the extract, antioxidants function as inhibitors of oxidation, effectively preventing the occurrence of harmful chain reactions at low levels. Consequently, they effectively eliminate the risks of pathological processes. The antioxidant properties of phenolic compounds discovered in medicinal plants have been well-documented. It has been reported that Cansjera rheedii leaves contain many active phytochemicals. Flavonoids, secondary metabolite compounds found in plants, play an important role in kidney physiology and promote renal protection.²⁸ The kidney safeguarding is attributed to quercetin as it effectively reduces inflammatory mediators The prevention of reactive oxygen species generation in the kidneys has been observed^29,30,31. In the current study, the HERC plant extract exhibits robust antioxidant activity in the prevention of kidney damage. Additionally, in silico docking studies are to be conducted to identify the specific active phytochemicals responsible for the nephroprotection against gentamicin-induced renal toxicity.

CONFLICT OF INTEREST:

The authors declare that they have no Conflict of Interest.

ACKNOWLEDGMENTS:

The authors would like to thank the Principal and Management Staff of B.V.V.S H.S.K of Pharmacy Bagalkote for providing the necessary facilities and their support for completing this research work.

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Received on 24.11.2023 Modified on 07.03.2024

Research J. Pharm. and Tech 2024; 17(8):3863-3868.

DOI: 10.52711/0974-360X.2024.00599