Ameliorative effect of Apodytes dimidiata on cisplatin-induced nephrotoxicity in Wistar rats

Abstract

Context Nutraceuticals possessing antioxidant potential have been used to alleviate side effects exerted by many chemotherapeutics, including cisplatin. Since Apodytes dimidiata E. Mey. Ex Arn. (Icacinaceae) shows antioxidant potential, it may possess significant chemoprotective effects.

Objectives The study investigated whether A. dimidiata could attenuate cisplatin-induced renal damage.

Materials and methods Nephrotoxicity was induced by cisplatin (single i.p., 16 mg/kg b wt.) in Wistar rats. Methanolic leaf extract of A. dimidiata (AMF) was administered at a dose of 250 mg/kg b. wt. orally for 5 consecutive days before/after cisplatin administration. Blood and renal parameters were analysed. Total phenolic and flavonoid content in AMF and its NO scavenging effect was determined.

Results Significant protective effect of AMF on cisplatin-induced nephrotoxicity was observed in pre-treated animals. The reduction of urea, creatinine and lipid peroxidation was 58.31%, 42.19% and 60%, respectively, and the increase in haemoglobin and leucocyte count was 28.25% and 42.91%, respectively. The increase calculated for GSH, GP_x, SOD and catalase was 35.64%, 18.14%, 74.42% and 35.46%, respectively. Tissue architecture of kidney was almost normal in AMF treated animals. The results were comparable to the standard drug, silymarin. AMF contained high level of polyphenols and flavonoids and was found to scavenge NO radicals (IC₅₀ 121.8 μg/mL).

Discussion and conclusion AMF can effectively counteract cisplatin mediated renal acute toxicity possibly by scavenging reactive oxygen and nitrogen species. Accordingly, the study suggests that AMF can ameliorate free radical-induced damage associated with chemotherapeutic drugs.

Keywords:

• Antioxidant activity
• nephroprotection
• oxidative stress
• renal failure

Introduction

Apodytes dimidiata E. Mey. Ex Arn. (Icacinaceae), a small bushy tree is seen in southern and eastern parts of Africa and different Asian countries along South and Central Sahyadris of India. The tree is valued in Zulu traditional medicine for various ailments. An infusion from the root bark is used as purgative to treat intestinal parasites (Gestner 1938; Bryant 1966; Hutchings et al. 1996) and the leaves are used in the treatment of ear inflammation (Watt & Breyer-Brandwijk 1962). Recently, a well-known anticancer alkaloid, camptothecin was isolated from the bark of this plant (Ramesha et al. 2013) and also from Fusarium solani (Mart) Sacc. (Nectriaceae), an endophytic fungus of A. dimidiata (Shweta et al. 2010). Six new saponins were reported from the leaves of A. dimidiata (Kenn et al. 2011). A study on the validation of antimycobacterial plants used by traditional healers in three districts of the Limpopo Province (South Africa) shows that A. dimidiata has antibacterial and antioxidant properties. The phytochemical screening of leaf acetone extract showed the presence of saponins, tannins, terpenes, steroids and flavonoids (Masoko & Nxumalo 2013). Moreover, in our previous studies, the antitumour (Divya et al. 2015) and chemopreventive (Divya et al. 2016) effects of A. dimidiata were revealed.

Free radical mediated toxicity is one of the major side effects of chemotherapeutic drugs. Cisplatin (CP-diamminedichloroplatinum II), a heavy metal complex, is a widely used antineoplastic drug for the treatment of several cancers. But, nephrotoxicity is reported as a major side effect conjoined with cisplatin treatment. Generation of oxidative stress and release of NO are considered to be the prime factors for cisplatin-induced nephrotoxicity (Pan et al. 2009). Superoxide (Davis et al. 2001), hydrogen peroxide (Kadikoylu et al. 2004) and hydroxyl (Shino et al. 2003) radicals increased by cisplatin, stimulates lipid peroxidation (Sadzuka et al. 1992) and results in the inhibition of antioxidant enzyme activities (Mora et al. 2003), which is responsible for nephrotoxicity (Amptoulach & Tsavaris 2011; Naqshbandi et al. 2012; Ognjanovic et al. 2012). To ameliorate these side effects, complementary agents of high antioxidant potential have been recently investigated (Monti & Yang 2005). Mansour et al. (2002) reported that antioxidants play an important role in attenuating nephrotoxicity mediated by oxidative stress.

Potential nutraceuticals such as natural antioxidants have been used to reduce side effects as well as to enhance the activities of chemotherapeutics (Zhang et al. 2010). Phenol and flavonoid class of compounds are natural antioxidants as they can quench reactive oxygen species efficiently (Korkina & Afanasev 1997). In the present study, an effort has been made to assess the protective effect of A. dimidiata on cisplatin-induced nephrotoxicity in rats. Moreover, the nitrogen scavenging activity and the total phenolic and flavonoid content in A. dimidiata are also evaluated.

Material and methods

Plant material

The leaves of Apodytes dimidiata were collected from Periya Tiger Reserve Forest, Wayanad District, Kerala, India (Altitude: 810 m, Geographical location: Lat. N 11°51'03.19″, Long. 75°48'05.54″) during the month of January 2014 and identified by Dr. Sujanapal P, Taxonomist, Kerala Forest Research Institute (KFRI) Peechi, Kerala. A voucher specimen (No. KFRI 28024) is kept at the Herbarium of KFRI.

Preparation of extract

Leaves of A. dimidiata were dried under shade and powdered using a mixer grinder. Approximately, 30 g of the powder was extracted separately with 250 mL of petroleum benzene, chloroform, acetone and methanol using Soxhlet for 24 h. Each extract was concentrated to dryness. The extract obtained in methanol was loaded to a column (600 mm × 30 mm) packed with 60–120 mesh silica gel and eluted successively by passing various solvents (150 mL each) of increasing polarities such as petroleum benzene, chloroform, acetone and methanol. The methanolic fraction (AMF) was used for studies.

Chemicals

Nitrobluetetrazolium (NBT), glutathione (GSH), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and riboflavin were purchased from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). Thiobarbituric acid (TBA) was purchased from Hi-Media Laboratories, Mumbai, India. Cisplatin was obtained from Kemoplat, Dabur Pharma Ltd., New Delhi, India. Silymarin was obtained from Krypton Pharmaceuticals, Mumbai. RPMI-1640 culture medium was purchased from Sigma Aldrich (St. Louis, MO) and FBS from PAN Biotech, Aidenbach, Germany. All other chemicals and reagents used were of analytical grade.

Animals

Wistar rats (120–150 g) used in the study were purchased from Small Animal Breeding Station, Kerala Veterinary and Animal Sciences University (KVASU), Thrissur, Kerala and were housed in well ventilated cages under controlled conditions of light and humidity and provided with normal mouse chow (Sai Durga Food and Feeds, Bangalore, India) and water ad libitum. The animal experiment in the study was carried out with the prior approval from Institutional Animal Ethics Committee by strictly following the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted by Animal Welfare Division, Government of India (No. 149/1999/CPCSEA).

Phytochemical screening and quantitative measurement of total phenolic and flavonoid contents

AMF was subjected to phytochemical screening by standard methods (Harbone 1973; Sofowora 1993; Evans et al. 2002). The total flavonoid content was analysed by colorimetric method (Chang et al. 2002). Briefly, the plant extract (0.5 mL) was mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminium chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water. The reaction mixture was allowed to stand at room temperature for 30 min and the absorbance of the reaction mixture was measured at 415 nm. A calibration curve was prepared by using quercetin at concentrations of 50 to 250 μg/mL in methanol. Flavonoid content was expressed as μg/mg quercetin equivalent (QE) of dry extract. Total phenolic content was determined by using Folin-Ciocalteu reagent (as modified by Ainsworth & Gillespie 2007). Gallic acid (20–100 μg/mL) was used as a reference standard for plotting calibration curve. A volume of 0.5 mL of the plant extract (100 μg/mL) was mixed with 2 mL of the Folin-Ciocalteu reagent (diluted 1:10 with de-ionized water) and were neutralized with 4 mL of sodium carbonate solution (7.5%, w/v). The reaction mixture was incubated at room temperature for 30 min with intermittent shaking and the absorbance of the resulting blue colour was measured at 765 nm. Total phenolic content was determined from the linear equation of a standard curve prepared with gallic acid. The content of total phenolic compounds is expressed as μg/mg gallic acid equivalent (GAE) of dry extract.

Experimental design

Male Wistar rats were divided into six groups of six animals each. Group 1 animals were kept as normal without any treatment. All animals in the other groups received cisplatin i.p. (16 mg/kg b. wt). Group 2 animals (untreated control) received no treatment, Group 3 was given propylene glycol (> 99% pure) and kept as vehicle control (200 μL), Group 4 animals were treated with standard drug silymarin at 100 mg/kg b. wt and groups 5 and 6 received AMF 250 mg/kg b. wt., respectively, by oral gavage for 5 days before and after cisplatin administration. All the mice in groups 2–6 were treated with single dose of cisplatin (16 mg/kg b. wt, i.p.) to induce renal damage (Somani et al. 2000). At the end of the experiment, the animals were sacrificed using ether anaesthesia and kidneys were removed and kept at −20 °C until homogenization.

Sample collection and biochemical analysis

Blood was collected directly from the heart in heparinized vials to determine the total WBC count (Chaudhari 2000) and haemoglobin content (Drabkin & Austin 1932). Blood was collected in non-heparinized vials to separate serum for the analysis of urea by Berthelot reaction method (Allain et al. 1974) and creatinine by Jaffe’s reaction method (Moss et al. 1975) using commercially available kits (Span Diagnostics, Surat, India).

Kidney was excised out, washed in ice-cold saline and a portion of tissue was fixed in 10% formalin for histopathological analysis. Kidney homogenates (10%, w/v) were prepared in Tris–HCl buffer (0.1 M, pH 7.4). The concentration of malondialdehyde (MDA) as an indicator of lipid peroxidation was determined according to a modified method of Ohkawa et al. (1979) based on the reaction with TBA. Superoxide dismutase (SOD) activity was carried out by the method of McCord and Fridovich (1969). The scavenging ability of the enzyme on superoxide anion radicals was determined by light-induced superoxide generation with riboflavin and subsequent reduction of nitrobluetetrazolium. Reduced GSH was measured by the method described by Moron et al. (1979). Glutathione peroxidase (GPx) was determined by the method described by Hafemann et al. (1974) by the degradation of hydrogen peroxide in the presence of GSH, thereby depleting it. The GSH remaining was measured using DTNB, which yields a coloured complex. Catalase (CAT) activity was determined by measuring the decomposition of hydrogen peroxide at 240 nm, according to the method of Aebi (1974). Furthermore, total protein was determined according to Lowry et al. (1951).

Nitric oxide radical inhibition assay

Macrophages were elicited by injecting 5% sodium caseinate intra-peritoneally in BALB/c mice, washed with PBS and resuspended in RPMI-1640 with 10% FCS. The cells were plated in 96-well culture plates and incubated for 2 h at 37 °C in a 5% CO₂ atmosphere. After incubation, non-adherent cells were removed and the adherent macrophages were incubated (2 × 10⁶ cells/well) in complete medium (RPMI-1640, 10% FCS, 100 μg/mL streptomycin and penicillin, 2 mM glutamine). Macrophages were cultured in the presence of various concentrations of AMF and the standard drug, ascorbic acid. After 24 h, plates were centrifuged and the supernatant was used for the estimation of NO by the Griess method (Stuehr & Nathan 1989). In brief, 50 μL of supernatant was mixed with 1% sulphanilamide/0.1% naphthylethylenediamine/2.5% H₃PO₄ and was incubated at room temperature for 10 min to form a chromophore. A typical blank/control solution containing the same solution mixture without plant extract was measured at 550 nm. The percentage inhibition was calculated according to the following equation: % inhibition = (1−A₁/A₀×100), where A_l is the absorbance of the extract and A₀ is the absorbance of the control. The concentration of sample required to inhibit 50% of nitric oxide radical (IC₅₀ value) was calculated by plotting the graph.

Statistical analysis

Data obtained in the experiment were expressed in terms of mean ± SEM. Statistical significance of data was assessed by the analysis of variance (one-way ANOVA) followed by a comparison between vehicle control and treated groups using ‘Tukey–Kramer’ multiple comparison test with Instat 3 software package (San Diego, CA). The significance level was set at p < 0.05.

Results

Phytochemical analysis of AMF

An estimated amount of total phenolic compounds in methanolic extract of A. dimidiata leaf was found to be 61.22 μg/mg GAE and the total flavonoid compounds in the same was 84.75 μg/mg QE. The other phytoconstituents evidenced by preliminary phytochemical screening were alkaloids, saponins, terpenes, carbohydrates, steroids, flavonoids, glycosides and tannins.

Effect of AMF on haematological parameters

Total WBC count was found to be significantly reduced in cisplatin alone treated animals. There was a proportionate increase in WBC (cells/mL × 10⁶) count with both pre- and post-treatment of AMF and the significance (p < 0.001) was same compared to that of vehicle control (Figure 1A). Similarly, cisplatin-induced decrease in haemoglobin level (Hb in g/dL) was improved to 11.98 ± 1.53 (p < 0.01) and 11.56 ± 1.73 (p < 0.05) with AMF pre- and post-treatment, respectively (Figure 1B).

Figure 1. Effect of AMF on total WBC count (A) and haemoglobin level (B) in cisplatin treated mice. The data obtained were represented as mean ± SEM (n = 6) and analysed using one-way ANOVA and group means were compared using the Tukey–Kramer multiple comparison test. The values are statistically different from vehicle control at p < 0.05^a, 0.01^b and 0.001^c and the values > 0.05 are considered to be non-significant.

Effect of AMF on biochemical parameters

Serum levels of creatinine (mg/dL) and urea (mg/dL) were increased in cisplatin alone treated group compared to normal group. The level of creatinine in cisplatin treated animals (5.42 ± 0.35) was significantly reduced to 3.26 ± 0.54 (p < 0.001) and 4.41 ± 0.25 (p < 0.001) in AMF pre- and post-treated groups, respectively (Figure 2A). Similarly, the elevated level of urea (139.21 ± 8.9) was significantly decreased to 51.56 ± 1.82 (p < 0.001) and 88.54 ± 2.56 (p < 0.001) in AMF pre-treated and post-treated groups, respectively (Figure 2B). The effect of AMF on the normalization of these kidney markers are comparable to that of silymarin (standard drug) treated animals.

Figure 2. Effect of AMF on serum creatinine (A) and urea (B) levels in cisplatin treated mice. The data were represented as mean ± SEM (n = 6) and analysed using one-way ANOVA and group means were compared using the Tukey–Kramer multiple comparison test. The values are statistically different from the control at p < 0.05^a, 0.01^b and 0.001^c and the values > 0.05 are considered to be non-significant.

Cisplatin-induced decline in the activity of SOD (0.58 ± 0.16 U/mg protein), GPx (21.98 ± 3.21 U/mg protein) and CAT (3.74 ± 0.29 U/mg protein) was found to be reversed to 1.92 ± 0.23 (p < 0.001), 27.89 ± 2.45 (p < 0.01) and 5.48 ± 0.47 (p < 0.001), respectively, by the pre-treatment of AMF and 1.1 ± 0.29 (p < 0.05), 22.41 ± 1.78 (non-significant) and 4.75 ± 0.43 (p < 0.01) by the post-treatment (Figure 3A–C). Similar observation was seen with GSH (nmol/mg protein) in which the depleted level (30.34 ± 1.04) was elevated to 45.76 ± 2.08 (p < 0.001) and 35.01 ± 2.32 (p < 0.01) by the pre- and post-treatment of AMF (Figure 3D). Also, a significant reduction in lipid peroxidation status was observed as inferred by the value of MDA (nmol/mg protein), 1.56 ± 0.24 (p < 0.001) and 2.34 ± 0.33 (p < 0.001) for the pre- and post-treated groups. In cisplatin treated animals, the MDA level was raised to 4.13 ± 0.59 (Figure 4).

Figure 3. Effect of AMF on SOD (A), GPx (B), CAT (C) activities and GSH (D) level in kidney tissue of cisplatin treated mice. The data obtained were represented as mean ± SEM (n = 6) and analysed using one-way ANOVA and group means were compared using the Tukey–Kramer multiple comparison test. The values are statistically different from the control at p < 0.05^a, 0.01^b and 0.001^c and the values >0.05 are considered to be non-significant.

Figure 4. Effect of AMF on lipid peroxidation in the kidney tissue of cisplatin treated mice. The data obtained were represented as mean ± SEM (n = 6) and analysed using one-way ANOVA and group means were compared using the Tukey–Kramer multiple comparison test. The values are statistically different from the control at p < 0.05^a, 0.01^b and 0.001^c and the values >0.05 are considered to be non-significant.

Effect of AMF on cisplatin-induced kidney damage

Histological details of kidney tissue from cisplatin and propylene glycol (vehicle control) treated animals showed a moderate to severe degree of damage. There was glomerular and tubular congestion with abnormal Bowman’s capsule, blood vessel constriction, vacuolation, necrosis and hemorrhagic areas. Inflammatory cells were also observed. Very few collections of lymphocytes were seen in the interstitial tissue. The histological features of both pre- and post-treatment groups showed minimal cellular damage in contrast to the control group. The AMF pre-treated group showed more recovery with almost normal glomerular and tubular arrangements, minimal blood vessel congestion and very few inflammatory cells (Figure 5).

Figure 5. Histopathology of mouse kidney. (A) normal (B) cisplatin alone (16 mg/kg); (C) cisplatin and silymarin (100 mg/kg); (D) cisplatin and AMF (250 mg/kg) treated mice. The slides were observed under light microscope at 400× magnification.

Scavenging effect of AMF on nitric oxide radicals

AMF exhibited potent nitric oxide scavenging activity with an IC₅₀ value of 121.8 μg/mL and the scavenging was found to be in a dose-dependent manner. The standard drug, ascorbic acid, exhibited IC₅₀ value at 68.9 μg/mL (Figure 6).

Figure 6. Nitric oxide (NO) radical scavenging effect of AMF. Ascorbic acid was used as a standard drug. Values are calculated as mean ± standard deviation of three independent replicates.

Discussion

Cisplatin is a chemotherapeutic drug currently used in the treatment of a wide range of cancers. However, the clinical usefulness of this drug is limited due to its side effects including nephrotoxicity. When cisplatin accumulates in renal epithelial cells, the GSH concentration and antioxidant enzymes activities become depleted which eventually leads to the hike in intracellular concentrations of ROS (Badary et al. 2005). Cisplatin also enhances Ca⁺⁺independent nitric oxide synthase activity leading to the increased production of nitric oxide (Srivastava et al. 1996). Among the approaches used to ameliorate or protect the cisplatin-induced nephrotoxicities, the most consistent effects have been observed with the use of antioxidant agents (Majid et al. 2012).

In the present study, the rats treated with single dose of cisplatin showed marked increase in serum creatinine and urea levels. The activity of various antioxidant enzymes like SOD, peroxidase and CAT in the kidney was found reduced. A concomitant increase in lipid peroxidation and depletion of GSH level along with disrupted tissue architecture clearly indicated significant nephrotoxicity by cisplatin administration.

Cisplatin-induced increase in urea and creatinine levels was found to be reduced by the oral administration of AMF. GSH depletion and associated lipid peroxidation was attenuated in these animals. Furthermore, the reduced activities of antioxidant enzymes by cisplatin were found restored by AMF to a considerable extent, indicating its ability to counteract the oxidative stress. Therefore, the nephroprotective effect of AMF observed in this study is likely to be mediated through its antioxidant property. These biochemical results were well corroborated by hisptopathological observations. This protective efficacy of AMF was comparable to that of standard drug, silymarin. Moreover, pre-treatment rather than the post-treatment with AMF exhibited better ameliorative effect. In pre-treatment, AMF might have improved the antioxidant status of the tissue environment and ultimately decreased the peroxidative damages.

The phytochemical screening of AMF had revealed the presence of various antioxidant constituents like saponins, tannins, steroids and appreciable quantities of polyphenols and flavonoids. Polyphenols and flavonoids are proved effective as ROS scavengers and metal chelators due to the presence of multiple hydroxyl groups (Bors et al. 1996). Renal content of peroxynitrite and nitric oxide is increased in cisplatin treated rats (Chirino et al. 2004), indicating the role of NO in nephrotoxicity. In this study, AMF inhibited NO generation in an in vitro system. Flavonoids which are considered effective scavenger of NO (Shutenko et al. 1999) were detected in the AMF. It is thus suggested that nephroprotective effect of A. dimidiata may be due to its polyphenol and flavonoid contents. Moreover, in our previous studies, the presence of an iridioid glycoside, genipin in the AMF fraction was detected while ascertaining the cytotoxic and antitumour properties of AMF (Divya et al. 2015). Drewes et al. (1996) had isolated genipin from the bark of A. dimidiata. Genipin has been reported to possess anti-inflammatory activity in air pouch oedema model in rat, by the inhibition of NO production (Koo et al. 2006). It is likely that NO scavenging ability of AMF may have a contributory role in nephroprotectivity. In an earlier study, NaF-induced imbalance in redox status was found to be regained by AMF administration in mice (Divya et al. 2016). Thus, the nitric oxide and other oxygen free radical scavenging phytoconstituents in AMF might be responsible for the nephroprotective effect. Further investigations are needed to identify and characterize the actual biomolecules in AMF responsible for its nephroprotective action.

Conclusion

AMF offers significant protection against cisplatin mediated nephrotoxicity primarily through its ROS and NO scavenging ability, suggesting its potential as an adjuvant in chemotherapy.

Funding information

The authors are thankful to Kerala State Council for Science Technology and Environment for providing the research fellowship (No 009-26/fshp/09/cste) to undertake this work.

Disclosure statement

The authors report no conflicts of interest.