Ficus benghalensis is a remarkable tree from India that sends down its branches and great number of shoots, which take root and become new trunks [1]. Its chemical constituent’s flavonoids leucoanthrocyanidin, leucoanthocyanin, friedelin, β sitosterol, querecitin-3-glactoside and rutin. Earlier, glucoside, 20-tetratriaconthene-2-one, 6-heptatriacontene-10-one, pentatriacontan-5-one, beta sitosterolalpha- D-glucose, and meso-inositol have been isolated from the bark of the F. benghalensis and it latex contains Caoytchoue (2.4%), Resin, Albumin, Cerin, sugar, and Malic acid [2] and used for treatment of neuralgia, rheumatism, lumbago, bruises, nasitis, gonorrhoea, inflammations, cracks of the sole and skin diseases [3] and in ayurveda for diarrhea, dysentery, and piles [4]. The extract of F. benghalensis was reported to inhibit insulinase activity from the liver and kidney and it was also found to inhibit the lipid peroxidation. F. benghalensis was traditionally used for the treatment of mehavikar or urinary disorders [5] but no scientific studies have been undertaken to verify these claims. Thus, the purpose of current study was to investigate whether oral administration of F. benghalensis latex extract has possible protective effect again^st cisplatin induced hypotension and renal impairment in wistar rats.

Phytochemical standardization

Phytochemical identification and standardization of FBLE performed by TLC Method and HPTLC (CAMAG Switzerland, Linomet 5, and Scanner 3, Win Cat Software) Mobile phase: Butanol: Formic acid: Water (7.5 ml: 1.5 ml: 1.0 ml). HPTLC analysis performed by use of various standard amino acid markers like glutamine, glycine, cysteine, methione, lysine, arginine etc., and extract in which one compound was identified on the extract track and their RF value 0.56 was similar to standard methionine marker. The methionine content of FBLE standardized that was found 0.842 ± 0.0364% of standard methionine.

Determination of total phenolic and flavonoid contents in FBLE

The total Phenolic and Flavonoid content of latex extract determined by method [6,7] respectively.

In vitro antioxidant activity

In vitro antioxidant studies of FBLE evaluated by Method DPPH [8] ferric chloride [9] phosphor-molybdenum [10] free radical scavenging.

Animals

Adult male Wistar rats (180-210 g) have an access to water and food ad libitum, and maintained under constant (25 ± 1°CAS), humidity (65 ± 10%) and a 12 h light/dark cycle. The experiment was carried out in accordance to the guidelines mentioned in the CPCSEA, and IAEC approved the experiment protocols (SVU/PH/IAEC/26.03.2010/02).

Acute toxicity study

Each group of Wistar rats fasted overnight prior to the experiment. Each group of rats fed FBLE dissolved in normal saline with increasing dose like 5, 50, 100, 200, 400, 1000 2000 mg/kg body weight. The animals observed continuously for 2 h and then every 2 h up to 24 and 72 h for gross behavior changes. So LD₅₀ cut off of the extract was 2000 mg/kg body weight. FBLE dose regimen prepared like 1/10^{th,/ and 1/5th of the respective LD₅₀ cut off values.}

Cisplatin-induced renal injury

Five groups of rats (n=6) used, in which 1^st group administered 5 ml/kg normal saline throughout the experiment for 16 days; 2^nd control group received FBLE 400 mg/kg per day for 16 days; 3^rd group (cisplatin treated) with single dose of cisplatin (5 mg/kg, i.p.) on 1^st day and keep animals up to 6 days; 4^th Group and 5^thGroup (Protective) FBLE (200 and 400 mg/kg, p.o. for 1^st to 10^thday and single dose of cisplatin (5 mg/ kg, i.p.) on 11^thday and keep animals up to 16 days [11].

On the 6^thday in cisplatin control and 16^thday in control, protective were measured blood pressure by help of student physiograph (instruments & chemicals PVT. LTD, Ambala, India) after then blood withdrawn from retro-orbital sinus of rats for biochemical estimation for serum urea and creatinine levels using diagnostic kit from Span Diagnostic, “Kolkata on chemical analyzer (Microlab 3000) and also dissected out the kidneys for estimation of In vivo antioxidant enzymes and histopathological works [12].”

In vivo Antioxidant activity

Rat kidneys homogenized and centrifuged at 10,000 rpm at 0ºC for 20 min. The supernatant used for estimation of antioxidant enzymes level by calorimetric method using spectrophotometer (Merck thermo spectronic, Model NO. UV-1, double beam), Glutathione reductase (GSH) estimated by method [13] Lipid peroxidation by thiobarbuturic acid-reactive substances (TBARS) methods [14] Superoxide dismutase (SOD) by method [15] Catalase (CAT) by colorimetric assay [16] and the sediment of the centrifuge used for estimation of the Na⁺K⁺ATPase by method [17] Ca²⁺ ATPase [18] Mg²⁺ ATPase [19].

Statistical analysis

Result were expressed as mean ± SEM, Statistical Analysis were performed with one way analysis of variance (ANOVA) followed by Dunnett’s test. P value less than <0.05 was considered significant.

FBLE has revealed presence of glycoside; alkaloids, tannin (Phenolic compound), Flavonoids, and methionine amino acid (Figures 1 and 2). Total Phenolic and flavonoids content had obtained 2.76 ± 0.84 mg GAE/g and 1.84 ± 0.5 mg QE/g extract respectively.

Figure 1: HPTLC chromatogram (a) standard methionine RF= 0.57; (b) HPTLC chromatogram extract RF=0.56.

Figure 2: Effect methanol Ficus benghalensis L. latex extract on average change body weight of various groups as compared to cisplatin treated group (3). Each group represents mean ± S.D. of six animals, **P<0.01,*P>0.05, **P>0.01, **P<0.01, **P>0.01 as compared to the cisplatin treated group.

In vitro antioxidant potential of FBLE was evaluated by scavenging effect of DPPH, ferric chloride, and phosphor-molybdenum. IC₅₀ values for DPPH, and phosphor-molybdenum were 28.63 ± 0.16 μg/ ml, and 31.84 ± 0.12 μg/ml respectively (Figures 3 and 4).

Figure 3: % DPPH radical scavenging activity of FBLE.

Figure 4: % Phosphor-molybdenum radical scavenging activity of FBLE.

Blood pressure of cisplatin-treated was decreased to 70 mmHg which is significantly increased 100 mmHg in protective groups.

The cisplatin-treated showed a significant increase urine volume, serum urea and creatinine levels, lipid peroxidation and decrease body wt. (Figure 2), GSH, SOD, CAT, Na⁺/K⁺ ATPase, Ca⁺⁺ ATPase, Mg⁺⁺ATPase of kidney (Tables 1-3), on the 6^thday as compared to the group I. They were significantly (p<0.01) recovered in protective regimen with treated dose at 200 and 400 mg/kg of FBLE.

Table 1: Effects of methanol extract of Ficusbenghalensis latexL. on the Urinary volume, Urea and Creatinine level in serum on 6th day after cisplatin administration.

Table 2: Effect ofmethanol extract of Ficus benghalensis latexL. on the lipid peroxidation and antioxidant enzymes of kidney on 6th day after cisplatin administration.

Table 3:  Effects of methanol extract of Ficus benghalensisL. latexon ATPase in kidney tissue of various groups on 6^th day after cisplatin administration.

Histopathological sections of the kidneys showed marked vasoconstriction, hyaline droplets, proinflammatory and tubular necrosis were observed cisplatin treated group III (Figure 5; Plates 1A- 1C) and in the protective regimen extract (200 and 400 mg/kg body wt., p.o.) reduced hyaline droplets, tubular dilation and recovery of tubular necrosis in which 400 mg/kg more effective reduction than 200 mg/kg (Figure 5; Plate 1D and 1E) respectively.

Figure 5: “Histopathology sections of kidney; Plate 1(A) Vehicle-treated, Plate 1 (B) FBLE treated, Plate 1 (C) Cisplatin-treated rats (5 mg/kg, 6 days), Plate 1 (D) Protective (200 mg/kg), and Plate 1 (E) Protective (400 mg/kg)”.

In the present study, cisplatin-induced renal impairment was evidenced by an increase in serum urea and creatinine and acute tubular necrosis. These changes observed on 6^thday after administration of a single dose 5 mg/kg cisplatin. FBLE normalized, raised serum urea, creatinine levels, lipid peroxidation and decreased blood pressure, GSH, SOD, CAT, Na⁺/K⁺ ATPase, Ca⁺⁺ ATPase, Mg⁺⁺ATPase of kidney. The histopathological report supported the biochemical findings.

The FBLE treated dose 400 mg/kg body weight was observed more significant than 200 mg/kg body wt.

According to previous findings, it was conformed that the single dose of cisplatin (5 mg/kg, i.p.) causes a significantly increase in two serum markers of the kidney function, viz. serum urea and creatinine [20]. Present study was revealed that significantly decrease the level of urea and creatinine in blood serum after treatment with FBLE that was indicate FBLE has nephroprotective activity. A relationship between oxidative stress and nephrotoxicity had well demonstrated in many experimental models Evidence point. The regulation up to normal blood pressure could be protective effect of renal impairment and activation sympathetic nerves system.

In vitro studies of FBLE evaluated for its good antioxidant potential revealed DPPH, ferric chloride, phosphor-molybdenum of free radical scavenging effect with lower IC₅₀ values. FBLE has been found to be a rich source of Caoytchoue (2.4%), Cerin, and Malic acid and present phytochemical data have been revealed tannin, Flavonoids, methionine. Yadav [21] also reported hepatoprotective effect of Ficus religiosa latex on cisplatin induced liver injury in Wistar rats.

Deegan et al. [22], also reported that nephrotoxicity, cytotoxicity and renal handling of a cisplatin methionine complex in male wistar rats. The present phytochemical screening data has observed methionine which was antagonized cisplatin nephrotoxicity.

Finally, it is concluded that FBLE could be an ameliorated cisplatin hypotension and nephrotoxicity.

Author declares no conflicts of interest.

This investigation was supported by MJRP University Jaipur.