2.6.1 Cell line

Human embryonic kidney-293 (HEK-293) cells were collected from the National Centre for Cell Science, Pune, India, and cultured in DMEM supplemented with 10% FBS and antibiotics and allowed to grow at 37 °C in a humidified incubator with 5% CO₂.

2.6.2 Protective effect of SBM against cisplatin induced nephrotoxicity and oxidative stress in HEK-293 cell line

After cytotoxicity estimation of SBM and cisplatin nephroprotective effect of SBM (3.9–500 µg/mL) was determined against cisplatin induced nephrotoxicity in HEK-293 cells. Briefly, cells were loaded on the 96-well plate and incubated with different concentration (3.9–500 µg/mL) for 24 h at 37 °C. Thereafter, the cells were treated with 100 µL of CP (13 µg/mL) and the plate was incubated for further 24 h. Then MTT reagent was mixed in each well and incubated for an additional 4 h at 37 °C. The formed blue formazan crystals were liquefied with the addition of 100 µL of solubilizing agent and absorbance recorded at 570 nm. Morphological changes in HEK-293 cells were examined before the addition of MTT reagent using EVOS XL Core Cell Imaging System (Kpemissi et al., 2019).

In Cellular antioxidant activity (CAA), intracellular formation of ROS was evaluated by using oxidation sensitive Dichloro-dihydro-fluorescein acetate (DCFH-DA) probes (Grauzdytė et al., 2018). Briefly, the pre-cultured HEK-293 cells in 96 well plates were treated with 100 µL of SBM (62.5–500 μg/mL) and the plate was incubated for 3 h. Thereafter, the washed cells were treated with 100 μL of DCFH-DA (10 μmol/L) after the treatment with cisplatin and fluorescence was recorded at excitation wavelength 485 nm and 530 nm.

2.7.1 Experimental animal

Wistar albino female rats (200–250 g) were utilized for in vivo studies and these rats obtained from Central Animal House of Hamdard University, approved (Approval Number: 1554) by Institutional Animal Ethics Committee (IAEC), Jamia Hamdard, New Delhi, India (Registration No.: 173/GO/RE/S/2000/CPCSEA).

2.7.2 CP-induced nephrotoxicity

The dose and time schedule of CP administration was adopted in this study were based on the pilot study done by Kumar et al. 2017. CP 7 mg/Kg body weight of through intraperitoneal (i.p.) route was used for the induction of nephrotoxicity. All the rats were divided into seven groups (n = 6 per group). Group 1 received normal saline (NS) and served as Control; Group II received CP (7 mg/kg/day, i.p.) and served as toxic control; Group III received SBM in low dose (LD-SBM, 32.1 mg/kg/day); Group IV received SBM in medium dose (MD-SBM, 64.2 mg/kg/day); Group V received SBM in high dose (HD-SBM, 128.4 mg/kg/day); Group VI received ascorbic acid (10 mg/kg/day, p.o.) and served as positive control; Group VII received α-ketoanalogue (10 mg/kg/day, p.o.) and served as positive control II. The dose of the sugar-free SBM formulation was calculated from the extractive value obtained with respect to the dose mentioned in Unani Pharmacopeia. Group II, III, IV, V, VI and VII received CP (7 mg/kg/day, i.p.) for the last two days of the experimental period i.e., 13th and 14th day. The body weight of all animals was measured pre and post treatment. At the end of the experiment, rats were sacrificed using ether anesthesia. Before scarification, blood samples were collected from retro-orbital and separate serum for biochemical analysis.

After the processing, the animals were sacrificed successively to collect kidney tissue from each animal to assess the antioxidant and histopathological analysis (Rezaee-Khorasany et al., 2020).

2.7.3 Evaluation of biomarkers in serum and urine

Collected sample of urine and serum was used for the investigation of different biochemical analysis. In kidney biomarkers analysis, blood urea (BU), creatinine (Cr), total protein (TP), albumin (Alb), globulin (Glb), total bilirubin (TB), direct bilirubin (DB), uric acid (UA), calcium (Ca), phosphorus (P), sodium (Na) and potassium (K) level was estimated on blood serum and urine. In hepatic biomarkers analysis, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) were performed on serum (Kpemissi et al., 2019).

2.7.4 Assessment of antioxidant status and oxidative stress biomarkers in the kidney

Collected kidney from each animal, homogenates (10% w/v) were made in tris–phosphate buffer (50 mM, pH 7.4) and then centrifuged at 1300 rpm for 10 min at 4 °C. The resultant supernatant were used to assess the glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and nitric oxide (KET900) levels using spectrophotometric assays (Kpemissi et al., 2019).

2.7.5 Determination of proinflammatory cytokines and caspase-3 in the kidney

Tumor necrosis factor-α (KET9007), interleukins-1β (KET900), caspase-3 levels (KET100992) were measured in kidney tissue homogenate as per the manufacturer protocol.

2.7.6 Histopathology

Histopathological studies of kidney tissues were conducted as per the described protocol using Olympus IX 71 research microscope (10×) (Sharma et al., 2017).

3.4.1 Effect of SBM on cytotoxicity of HEK-293 cells

The results showed 500 μg/mL and 13 μg/mL cytotoxic concentrations of SBM and cisplatin. The effect of SBM on cell viability was found in a dose-dependent manner. The graph of cytotoxicity assay of SBM and Ascorbic acid are summarized in Fig. 2a.

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Fig. 2

(a) Effect of sugar-free SBM and ascorbic acid on cell viability in HEK-293 cell line. Data are expressed as mean ± SEM (n = 3). One-way ANOVA followed by Tukey's multiple comparisons test. Compared to normal control group: The statistical significance level was expressed at *P < 0.05, **P < 0.01, ***P < 0.001; ns p > 0.5. (b) Nephroprotective potential of SBM on HEK-293 cell line using CP as toxicant. Data are expressed as mean ± SEM (n = 3). One-way ANOVA followed by Tukey's multiple comparisons test. Compared to normal control group: ## P < 0.01, ### P < 0.001; Compared to CP control group: The statistical significance level was expressed at *P < 0.05, **P < 0.01, ***P < 0.001; ns p > 0.5. 2(c) Fig. 6 Effect of SBM on CP- induced ROS production in HEK-293 cell line, the intracellular ROS production was quantified, expressed as percentage of control and show as mean ± SEM (n = 3). Compared to normal control group: ### P < 0.001; Compared to CP control group: The statistical significance level was expressed at **P < 0.01, ***P < 0.001; ns p > 0.5.

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3.4.2 Nephroprotective effect of SBM against CP-induced toxicity in HEK-293 cells

The nephroprotective effect of the sugar-free SBM was evaluated against CP-induced cytotoxicity in HEK-293 cells. The resulted data showed that CP treatment significantly (P < 0.001) reduced the cell viability which was reversed after the treatment with SBM. SBM was accompanied with 76.85 ± 1.50% viability at 500 µg/mL against the reduced viability of HEK-293 with 41.20 ± 0.05% by CP. Although the protective effect of SBM against the CP toxicity was found in a dose-dependent manner while no significant changes were observed compared to Ascorbic acid (Fig. 2b).

3.4.3 Cellular antioxidant potential of SBM

In order to evaluate the protective actions of SBM against CP-induced intracellular ROS production in HEK-293 cells, DCFH-DA fluorescent probe was used to measure intracellular ROS level in HEK-293 cells. The experimental findings reveal that level of ROS in HEK-293 cells was significantly increased with incubation of CP compared with the control group (Fig. 2c). Treatment with SBM at different concentrations (150–500 µg/mL) significantly decreases CP-induced ROS production. Ascorbic acid showed no significant difference compared to SBM. (Fig. 2c)

3.4.4 Effect of CP on morphology of HEK-293 cells

Morphological changes in HEK-293 cell lines have been assessed after induction of nephrotoxicity by CP and the results were observed as marked changes in the morphology of HEK-293 cells. The morphological change was assessed by phase-contrast microscopy as shown in Fig. 3.

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Fig. 3

Control cells (a) that shows a small, polygonal-shaped and epithelial-like morphology. In contrast, cells exposed to CP (b) expressing complete distorted architect as compared to control with loss to grow as epithelium-like monolayer and even with condensed intracellular parts and almost all the cells appeared with the spherical shape or rounded phenotype. (c-f) displays treatment groups of cells with SBM, at different concentration (500 C, 125 D, 31.25 E and 7.81 F μg/mL). After the treatment with SBM, figure C-D showing normal architect of cells with least cells death as compared to control cells whereas figure (e and f) showing significant changes in architect of cells with rounded phenotype. Figure K-N displays treatment groups of cells with ascorbic acid at different concentration (500 K, 125 L, 31.25 M and 7.81 N μg/mL). After the treatment with ascorbic acid, figure (g and h) showing normal architect of cells with least cells death as compared to control cells whereas figure (i and j) showing reduced number of cells but no significant changes in architect of cells with least round phenotype.

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3.5.1 Effect of SBM on serum biochemical markers

The resulted data reveals that two days of treatment with cisplatin causes significant changes in biomarkers of the kidney and liver. In contrast, serum levels of creatinine, urea and uric acid in the CP group were significantly increased (P < 0.05) compared to the normal control group, while the levels of total protein (TP), blood urea nitrogen (BUN), albumin (Alb), globulin (Glb), direct bilirubin, total bilirubin Table 2 and electrolytes (calcium, phosphorus, and Magnesium), were significantly (P < 0.05) decreased Table 3. Similarly, electrolytes such as sodium and potassium levels were found as significantly increased (P < 0.05) in CP group as compared to the normal control group. In addition, the serum levels of AST, ALT and ALP enzymes activity were also significantly (P < 0.05) increased in the CP group compared to the normal control. These altered levels of renal function parameters confirmed the induction of nephrotoxicity. However, the treatment with all three doses of SBM resulted in significant (P < 0.05) amelioration of renal and liver biomarkers.

3.5.2 Effect of SBM on urine biochemical markers

Administration of CP resulted in significant (p < 0.05) alteration in kidney function urine biomarkers such as creatinine, urea, uric acid and electrolytes compared to the control group. Treatment of all three doses of SBM exhibited significant (p < 0.05) normalization of all the kidney function parameters as compared to CP group (Table 4).

3.5.3 Effect on antioxidant oxidative stress parameter

Rat Kidney treated with CP had significantly higher (p < 0.05) MDA and NO levels, while SOD, CAT, GPx and GSH activities were significantly lower compared to the control group. Furthermore, pre-oral administration of all three doses of SBM, ascorbic and α-keto-analogue increase CAT, GPx, GSH and SOD (p < 0.05) level compare with CP group. Whereas, the treatment of ascorbic acid, α-ketoanalogue and all three doses of SBM significantly (p < 0.05) decrease the level of MDA and NO (Fig. 4a–f).

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Fig. 4

Effect of SBM on GPx [a], GSH [b], CAT [c], SOD [d], MDA [e] and NO [f] in CP-induced nephrotoxicity. Data are expressed as mean ± SEM (n = 6). One-way ANOVA followed by Tukey's multiple comparisons test. Compared to normal control group: ### P < 0.001; Compared to CP control group: The statistical significance level was expressed at *P < 0.05, **P < 0.01, ***P < 0.001; ns p > 0.5.

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3.5.4 Effect on inflammatory markers in kidney

CP treated rat showed significant (p < 0.05) higher levels of TNF-α, IL-1β and caspase-3 as compared with the normal control group. However, SBM potentially ameliorates the CP induces inflammatory stress. While the group co-administered ascorbic acid and α-ketoanalogue showed significant (p < 0.05) amelioration in oxidative stress induced by CP (Fig. 5a–c).

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Fig. 5

Effect of SBM on TNF-α [a], IL-1β [b], and Caspase-3 [c], in CP-induced nephrotoxicity. Data are expressed as mean ± SEM (n = 6). One-way ANOVA followed by Tukey's multiple comparisons test. Compared to normal control group: ### P < 0.001; Compared to CP control group: The statistical significance level was expressed at *P < 0.05, **P < 0.01, ***P < 0.001; ns p > 0.5.

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3.5.5 Effect of SBM on renal histological changes in Wistar albino rats

After administration of SBM, the altered renal morphology was found to be significantly restored to normal. The morphology of Bowman's capsule, proximal tubule, and distal tubule de-structured brush border cells and epithelial cells drastically changed after two days of cisplatin therapy. SBM, ascorbic acid and α-ketoanalogue restored the kidney architecture against CP group (Fig. 6).

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Fig. 6

Histopathological study of renal tissues of experimental animals (n = 6). No changes were noticed in the normal control group (a); The kidney samples of the CP (toxic control group) showed periglomerular mild infiltrate in inflammatory cells of few glomeruli. (b); The renal treated with low dose of SBM cortex showed numerous normal glomeruli having normal capillary loops, vascular endothelial cell swelling, mild interstitial congestion and edema, and multifocal mixed inflammatory infiltrates (c); kidney sample treated from medium dose of SBM, cortex showed numerous normal glomeruli having normal capillary loops, mesangial cells and mesangial matrix deposition is normal and mild infiltrate in inflammatory cells (d); The renal treated with high dose of SBM showed normal glomeruli, mesangial cells and mesangial matrix deposition is normal. (e); The kidney samples of ascorbic acid group cortex showed numerous normal glomeruli having normal capillary loops, mesangial cells and mesangial matrix deposition is normal. Proximal (f); The kidney section of α-ketoanalogue cortex showed numerous normal glomeruli having normal capillary loops, mesangial cells are not increased and mesangial matrix deposition is normal. Many glomeruli showed periglomerular mild infiltrate in inflammatory cells. Focal areas in cortex (near PCT) showed mild inflammatory cells infiltrate (g).

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