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ORIGINAL ARTICLE
Year : 2013  |  Volume : 9  |  Issue : 2  |  Page : 230-234

Ayurvedic formulations ameliorate cisplatin-induced nephrotoxicity: Preclinical studies on Brahma Rasayana and Chyavanaprash


Deptartment of Radiation Biology, Pushpagiri Institute of Medical Sciences and Research Centre, Thiruvalla, Kerala, India

Date of Web Publication13-Jun-2013

Correspondence Address:
Cherupally Krishnan Krishnan Nair
Deptartment of Radiation Biology, Pushpagiri Institute of Medical Sciences and Research Centre, Thiruvalla - 689 101, Kerala
India
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Source of Support: Board of Research in Nuclear Sciences, Department of Atomic Energy., Conflict of Interest: None


DOI: 10.4103/0973-1482.113363

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 > Abstract 

Aim of Study: To explore the ability of two Ayurvedic formulations, Brahma Rasayana (BRM) and Chyavanaprash (CHM) in alleviating Cisplatin (Cis-dichlorodiammineplatinum [II] CDDP) induced acute nephrotoxicity.
Materials and Methods: Swiss albino mice were administered with CDDP (12 mg/kg, i.p) and two doses of BRM or CHM (1 and 2 g/kg). Various antioxidant parameters in the kidney as well as release of marker enzymes in the serum were assayed. Histology of the kidney was also performed to check for CDDP induced damages.
Results: Administration of either BRM or CHM (1 and 2 g/kg) maintained the antioxidant status in the kidney thereby preventing tissue damage as well as the release of marker enzymes. CDDP induced variation of renal architecture was also prevented by BRM and CHM administration.
Conclusion: BRM and CHM administration could prevent CDDP induced acute renal toxicity.

Keywords: Antioxidant status, Brahma Rasayana, Chyavanaprash, cisplatin, nephrotoxicity


How to cite this article:
Menon A, Krishnan Nair CK. Ayurvedic formulations ameliorate cisplatin-induced nephrotoxicity: Preclinical studies on Brahma Rasayana and Chyavanaprash. J Can Res Ther 2013;9:230-4

How to cite this URL:
Menon A, Krishnan Nair CK. Ayurvedic formulations ameliorate cisplatin-induced nephrotoxicity: Preclinical studies on Brahma Rasayana and Chyavanaprash. J Can Res Ther [serial online] 2013 [cited 2019 Nov 14];9:230-4. Available from: http://www.cancerjournal.net/text.asp?2013/9/2/230/113363


 > Introduction Top


Cisplatin (Cis-dichlorodiammineplatinum [II] CDDP) represents a class of antineoplastic drugs containing a heavy metal, platinum. It is effective against several human tumors, e.g. testis, ovary, head, neck and lung. [1],[2] Though high doses of CDDP is preferred for tumour therapy, [3] its optimal clinical use is limited due to the numerous side effects it produces like nephrotoxicity, neurotoxicity, bone marrow toxicity, gastrointestinal toxicity and ototoxicity. [4],[5],[6] Among these, nephrotoxicity is the major side effect of CDDP since kidney accumulates cisplatin to a greater degree than other organs and is the major route for its excretion. [7],[8]

Recent evidences suggest the involvement of free radicals, depletion of GSH and other cellular antioxidant enzymes, in the induction of nephrotoxicity by CDDP. [9],[10],[11] Since free radicals bring out the basic mechanism of CDDP induced nephrotoxicity, it is rational to consider antioxidants as therapeutics to mitigate its toxicity. Various studies have reported the use of antioxidants in mitigating nephrotoxicity caused by CDDP. [12],[13],[14]

Ayurveda, the Indian holistic healthcare system of traditional medicines made using natural ingredients [15] have several potent antioxidant formulations. Two such formulations Brahma Rasayana (BRM) and Chyavanaprash (CHM) are evaluated in the present study for their nephroprotective ability since several ingredients of BRM and CHM are proved to be potent antioxidants. [16],[17] These formulations have shown promise in alleviating various free radicals induced physiological conditions. [18],[19] Also these are reported to confer tissue regeneration capability [20] and antitumour activity. [21],[22],[23] These findings have encouraged us to further investigate its potential during cancer therapy. As an initial step, the present study envisages to find out the ability of these Ayurvedic formulations in reducing nephrotoxicity induced by CDDP, which is a widely used chemotherapeutic agent, in mice model.


 > Materials and Methods Top


Chemical

Brahma Rasayana and Chyavanaprash, the two rasayana formulations prepared according to Sahasrayogam [24] were from reputed manufacturers available locally. All the other chemicals and reagents used in this study were of analytical grade.

Animals

Swiss albino mice of 8-10 weeks old, weighing 22-25 g was obtained from the Small Animal Breeding Section (SABS), Kerala Agricultural University, Mannuthy, Thrissur, Kerala. They were kept under standard conditions of temperature and humidity in the Centre's Animal House Facility. The animals were provided with standard mouse chow (Sai Durga Feeds and Foods, Bangalore, India) and water ad libitum. All animal experiments in this study were carried out with the prior approval of the Institutional Animal Ethics Committee (IAEC) and were conducted strictly adhering to the guidelines of Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted by the Animal Welfare Division of Government of India.

Experimental design

Animals were randomly divided into 6 groups of five each as detailed below. Group I was kept as the untreated control and all the other groups received CDDP (12 mg/kg, i.p). Group III and Group IV were administered with BRM or CHM (2 g/kg, p.o) after 1 hour of CDDP injection, i.p.

Untreated control.

CDDP (12 mg/kg, i.p).
CDDP (12 mg/kg, i.p) + BRM (1 g/kg, p.o).
CDDP (12 mg/kg, i.p) + BRM (2 g/kg, p.o).
CDDP (12 mg/kg, i.p) + CHM (1 g/kg, p.o).
CDDP (12 mg/kg, i.p) + CHM (2 g/kg, p.o).

Assessment of nephrotoxicity

72 hours after CDDP treatment, blood was collected by cardiac puncture and serum was separated for biochemical analysis. Kidney was excised and washed with ice-cold phosphate buffered saline (PBS) and homogenates 10% (w/v) were prepared in PBS. Serum creatinine level was determined by alkaline picric acid method and serum urea level was determined by diacetylmonoxime (DAM) reagent (Agappe Diagnostic Pvt. Ltd.; Ernakulam, Kerala, India). Level of GSH was assayed by the method of Moron (1974), [25] based on the reaction with DTNB. GPx activity was measured based on the method of Hafeman (1974), [26] based on the degradation of H 2 O 2 . Activity of SOD was measured by NBT reduction method of Mc Cord and Fridovich (1969). [27] Protein levels in the tissue were measured by following the method of Lowry (1951). [28] Levels in peroxidation of membrane lipids were done based on the method of Buege and Aust (1987). [29] For histopathological studies kidney was fixed in 10% formalin solution immediately after sacrifice, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Five-micron-thick sections made using a microtome were mounted on glass slides, dewaxed, rehydrated with distilled water, and stained with hematoxylin and eosin and mounted in DPX. [30] The slides were observed in light microscope under oil immersion microscope (100 X) and photographed.

Statistical analysis

The results are presented as Mean ± SD of the studied group. Statistical analyses of the results were performed using ANOVA with Tukey-Kramer multiple comparisons test.


 > Results Top


Antioxidant status

[Table 1] gives the cellular antioxidant levels (GSH, GPx and SOD). The results indicated that a single dose of CDDP (12 mg/kg) significantly decreased the tissue antioxidants. Oral administration of BRM or CHM not only helped in maintaining these antioxidants at normal levels, but also improved the total antioxidant status when compared to the control untreated animals.
Table 1: Effect of BRM or CHM on antioxidants level in CDDP induced nephrotoxicity in mice. Values are expressed as mean ± SD (n = 5)

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Peroxidation of membrane lipids

As presented in [Figure 1], CDDP (12 mg/kg) injection resulted in an increase in the levels of peroxidation of membrane lipids from 4.78 ± 1.56 to 8.34 ± 1.98 nMoles/mg protein. Administration of BRM or CHM helped in reducing the extent of peroxidation. BRM administration at a dose of 1 g/kg and 2 g/kg restored the level of peroxidation to 2.95 ± 1.17 and 2.15 ± 0.32 respectively. CHM administration at a dose of 1 g/kg and 2 g/kg restored the level of peroxidation to 3.01 ± 1.02 and 2.25 ± 0.43 respectively.
Figure 1: Effect of BRM or CHM on peroxidation of membrane lipids in CDDP induced nephrotoxicity in mice. Values are expressed as mean ± SD (n = 5). ***indicates 'P < 0.001' when compared with respective controls

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Serum biochemical parameters

The levels of various serum parameters presented in [Table 2] suggest that CDDP (12 mg/kg) administration increased these enzyme levels while the administration of BRM or CHM prevented the increase in the serum enzyme levels following CDDP treatment.
Table 2: Effect of BRM or CHM on serum marker enzymes in CDDP induced nephrotoxicity in mice. Values are expressed as mean ± SD (n = 5)

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Morphological study

Histopathological investigation showed that, in CDDP (12 mg/kg) treated mice kidney there is a decreased cellularity of the glomeruli and edema of the lining of epithelial cells in the renal tubules. More over the nuclei of the lining cells show vacoulation. The interstitial tissue also showed oedema as can be evident from [Figure 2]b. The renal tissues of CDDP treated mice when administered with BRM or CHM after the CDDP treatment, showed near normal architecture with normal glomerular, renal tubules and interstitial tissue appearance [Figure 2]c-f.
Figure 2: Effect of BRM and CHM various doses (1 and 2 g/kg) on CDDP induced renal damage in mice (× 40)

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 > Discussion Top


There are 2 suggested mechanisms by which CDDP inflicts nephrotoxicity. [31] One mechanism is by the cleavage of GSH by the enzyme gamma glutamyl transpeptidase (GGT) thereby releasing cysteinyl-glycine, which is further cleaved into cysteine and glycine by diaminopeptidase N. [32] Depletion of tissue GSH is a major factor which weakens the cell's defence against ROS mediated peroxidative cell injury. [33]

The other mechanism of renal toxicity is due to the formation of nephrotoxic metabolites of CDDP through a GSH-conjugate intermediate, a majority of which occurs in the kidney. [34] It has also been shown that inhibition of GSH reduces the toxicity of CDDP. [35],[36]

CDDP elicits its action against tumour cells by the activation of apoptotic pathways. [37] In recent years, kidney tubular apoptosis has been identified as a common pathway in response to cellular stress applied at intensity below the threshold for necrosis, [38] and this holds true in case of CDDP toxicity also. [39]

Cisplatin is known to accumulate in mitochondria of renal epithelial cells and induces ROS production, resulting in reduction of antioxidant status of kidney along with peroxidation of membrane lipids. [40],[41] Various antioxidant molecules have promise in mitigating the nephrotoxicity induced by CDDP. [42],[43],[44] The present work on the attempt to find the ability of two antioxidant Ayurvedic formulations in mitigating CDDP induced nephrotoxicity have produced interesting results, since 2 over the counter formulations are able to mitigate the adverse effects of a potent anticancer drug. These formulations have been reported to have antioxidant activity under in vitro conditions and also under in vivo conditions in various tissues, following consumption. [19] Administration of BRM or CHM prevented the CDDP induced nephrotoxicity by restoring the total antioxidant content. BRM and CHM are formulations used in 'rasayana therapy' in Ayurveda, which is a dedicated treatment modality for immune promotive, antidegenerative and rejuvenative health care. [45] The capacity to repair and regenerate damaged tissue, may also have contributed to the nephroprotective property of these formulations.

In the present study, a single dose of CDDP (12 mg/kg) induced nephrotoxicity was manifested biochemically by the increase in the levels of serum creatinine and urea. These results were consistent with previous studies reported by other investigators. [46],[47] Administration of BRM and CHM prevented the alterations in renal haemodynamics by CDDP administration.

The present study convincingly proved that BRM or CHM administration prevents the nephrotoxic effect of CDDP. Screening the vast repertoire of Ayurveda can yield in the discovery of formulations which can be useful in preventing the side effects of chemotherapeutic drugs. Further studies are to be conducted to know whether these formulations can offer preferential protection to normal tissues without affecting the antineoplastic activity of CDDP.


 > Acknowledgement Top


The authors thank Board of Research in Nuclear Sciences, Department of Atomic Energy for the financial support.

 
 > References Top

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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]


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