|Year : 2014 | Volume
| Issue : 3 | Page : 298-302
Ameliorative effect of Elaeocarpus ganitrus on gentamicin-induced nephrotoxicity in rats
Rahul Motiram Kakalij, Chaitanya P Alla, Rahul P Kshirsagar, Boyina Hemanth Kumar, Sumeet S Mutha, Prakash Vamanrao Diwan
Department of Pharmacology, School of Pharmacy, Anurag Group of Institutions, Venkatapur, Ghatkesar, Rangareddy, Hyderabad, Andhra Pradesh, India
|Date of Submission||12-Dec-2013|
|Date of Decision||31-Jan-2014|
|Date of Acceptance||21-Mar-2014|
|Date of Web Publication||9-May-2014|
Prakash Vamanrao Diwan
Department of Pharmacology, School of Pharmacy, Anurag Group of Institutions, Venkatapur, Ghatkesar, Rangareddy, Hyderabad, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Objectives: The present study was designed to evaluate the ameliorative effect of Elaeocarpus ganitrus on gentamicin (GM)-induced nephrotoxicity in rats.
Materials and Methods: E. ganitrus (100, 200, and 400 mg/kg body weight) was administered orally to male Wistar rats. GM (100 mg/kg) was used to induce nephrotoxicity. Study parameters include serum albumin, creatinine, blood urea nitrogen (BUN), uric acid, creatinine, and albuminuria. Total protein in serum, antioxidant enzymes activities, phagocytic index, and neutrophil adhesion assays were performed to determine oxidative stress and immunomodulatory action of E. ganitrus.
Results: The results revealed that coadministration of E. ganitrus significantly reduced the elevated level of serum creatinine, BUN, uric acid, and albuminuria with considerable increase in the serum albumin and urine creatinine. Furthermore, E. ganitrus noticeably increased serum total protein and antioxidant enzyme levels with significant alteration in phagocytic index and neutrophil adhesion assay when compared with GM-treated group in a dose-dependent manner.
Conclusion: The present study revealed that ethanolic extract of E. ganitrus seeds has immunomodulatory and nephroprotective activity.
Keywords: Elaeocarpus ganitrus , gentamicin, immunomodulatory, nephroprotective
|How to cite this article:|
Kakalij RM, Alla CP, Kshirsagar RP, Kumar BH, Mutha SS, Diwan PV. Ameliorative effect of Elaeocarpus ganitrus on gentamicin-induced nephrotoxicity in rats. Indian J Pharmacol 2014;46:298-302
|How to cite this URL:|
Kakalij RM, Alla CP, Kshirsagar RP, Kumar BH, Mutha SS, Diwan PV. Ameliorative effect of Elaeocarpus ganitrus on gentamicin-induced nephrotoxicity in rats. Indian J Pharmacol [serial online] 2014 [cited 2021 Oct 20];46:298-302. Available from: https://www.ijp-online.com/text.asp?2014/46/3/298/132163
| » Introduction|| |
Nephrotoxicity can be defined as renal disease or dysfunction that arises as a direct or indirect result of exposure to medicines and industrial or environmental chemicals. Gentamicin (GM) is an amino glycoside antibiotic that is very effective on life-threatening Gram-negative infections in humans and animals.  Unfortunately, 30% of patients treated with GM for more than 7 days show some signs of nephrotoxicity.  It has been reported that GM-induced nephrotoxicity characterized by direct tubular necrosis that is localized mainly in the proximal tubule is followed by renal failure with an increase plasma creatinine, urea,  and reactive oxygen species (ROS) generation in kidneys. , A considerable number of studies have demonstrated that various crude agents, including garlic,  arabic gum,  lycopene,  Spirulina platensis,  and others can prevent GM-induced renal damage.
Elaeocarpus ganitrus belongs to the family Elaeocarpeaceae found in tropical and subtropical areas. Traditionally, Rudraksha seeds have been used for the treatment of various ailments like stress, anxiety, depression, nerve pain, epilepsy, hypertension, arthritis, and liver diseases. E. ganitrus seed contains indolizidine type of alkaloids, which display a wide range of biological activities and are subject of various synthetic studies. It also contains minerals, flavanoids, vitamins, tannins, steroids, glycosides, and carbohydrates. The main compounds are ellagic acid, gallic acid, quercetin, elaeocarpidine, elaeocarpine, elaeocarpiline, and rudrakine.  Recently many therapeutic effects of E. ganitrus extracts have been documented, including antihypertensive,  antioxidant,  anti-inflammatory,  antidiuretic,  central analgesic,  antifungal,  and antimicrobial  effects in experimental studies. A comprehensive literature survey revealed that there are no scientific studies carried out regarding E. ganitrus in GM-induced nephrotoxicity in the aspects with relation to immune regulation and free radical scavenging property. Hence, the present study is focused to evaluate the nephroprotective, antioxidant, and anti-inflammatory potential of the E. ganitrus against GM-induced nephrotoxicity in Wistar rats.
| » Materials and Methods|| |
GM (Nitin Lifesciences Ltd, Karnal, India) and methylprednisolone (MP) (Pfizer Limited) were purchased from the local market. All other chemicals and reagents used were of analytical grade.
Male Wistar rats, procured from commercial breeder, were kept for a week for acclimatization under environmentally controlled conditions with free access to standard food and water. Rats weighing 150-250 g were carried out according to the guidelines and approval of Institutional Animal Ethics Committee, protocol no I/IAEC/009/2013/WR- 36.
Collection and Authentication of Plant Material
The E. ganitrus seeds were collected from Rudraksha Ayurvedic clinic, Hyderabad, seeds authenticated by Dr. V. Krishna Reddy, Assistant Professor, Department of Botany, Kakatiya University, Warangal, Andhra Pradesh, India.
Preparation of Extracts
Dried seeds of E. ganitrus were mechanically powder and sieved. The powder was extracted with 90% ethanol, concentrated under vacuum at temperature 60ºC, and dried to get the powdered form of the extract, and it was stored in tightly closed glass bottle in refrigerator until use.
Preliminary Phytochemical Screening
Preliminary phytochemical screening revealed the presence of phytosterols, fats, alkaloids, glycosides, tannins, proteins, carbohydrates, and flavonoids in the extract. 
Acute Toxicity Studies
The acute oral toxicity study was carried out as per guideline 423 set by the Organization for Economic Cooperation and Development received from Committee for the Purpose of Control and Supervision of Experiments on Animals. Lethal dose 50 value of E. ganitrus is not more than 5 g/kg body weight. 
Induction of Disease and Experimental Design
Animals were randomly divided into six groups of six animals each. Group I treated with vehicle (CMC, p. o.) was kept as normal. Group II was injected with GM (100 mg/kg, i. p.) for 6 days. Group III, IV, and V were administered E. ganitrus 100, 200, and 400 mg/kg, p. o. body weight along with GM, respectively, for 6 days. Group VI was administered MP (12.5 mg/kg, i. p.) for 6 days along with GM. After 24 h from last injection, blood samples were collected by retro-orbital plexus of rats. Blood samples were centrifuged to get serum for biochemical estimations. Kidneys were quickly removed for antioxidant and histopathological assessments.
Renal Function Tests
Urine and serum concentrations of albumin and uric acid were quantitatively determined using albumin and uric acid kits (ERBA, Mumbai). Urine creatinine was measured using an assay kit (ACCUREX biomedical Pvt. Ltd, Thane). Concentrations of blood urea nitrogen (BUN) were determined using BUN kit from BIOSYSTEMS. All the assays were performed in duplicate according to the manufacturer's instructions.
Determination of Total Protein
Tissue protein was estimated using bovine serum albumin as standard and was measured by the method described by Lowry et al. 
Antioxidant Enzyme Activity
Kidney homogenate (10%w/v) were prepared in phosphate-buffered saline (PBS) and supernatant was used for the estimation of super oxide dismutase (SOD) by pyrogallol oxidation method.  Glutathione (GSH) levels were measured spectrometrically using DTNB as standard. Catalase activity was determined from rate of decomposition of H 2 O 2 method. 
Neutrophil adhesion assay was done by the method described by Wilkonson.  Phagocytic index was determined by the method described by Gonda. 
Histopathological sections of kidney from all the treated groups were evaluated using light microscopy. For this the tissues were fixed in 10% formalin, embedded in paraffin, sectioned at 5mM, and stained with hematoxylin-eosin.
Results are given as mean ± standard error of mean (SEM). Data were analyzed using one-way analysis of variance (ANOVA) followed by Newman-Keuls test. The statistical significance of difference was taken as P < 0.05.
| » Results|| |
Renal Function Tests
Acute nephrotoxicity occurred in the group II due to GM injections during 6 days leads to increase level of serum creatinine, BUN, uric acid, and albumin in urine with statistical significance (P < 0.001) as compared with group I shown in the [Table 1]. The elevated serum creatinine, BUN, uric acid, and urine albumin levels were significantly (P < 0.05) reduced in groups III, IV, and V when compared with group II. However, differences in serum creatinine, BUN, uric acid, and albumin in urine were significantly (P < 0.05) different in groups III, IV, and V when compared with the group I that is the dose-dependent activity of E. ganitrus. In group VI there was a significant decrease (P < 0.05) in serum creatinine, BUN, and uric acid and increase in serum albumin and urine creatinine levels compared with group II.
Effect of E. ganitrus on total protein
GM caused decrease in total protein levels with (P < 0.001) significance in group II when compared with group I. In groups III, IV, and V there is elevated total protein levels with (P < 0.001) significance compared with group II. Group VI showed significant (P < 0.05) increase in total protein levels compared with group II.
Effect of E. ganitrus on renal oxidative stress biomarkers
In group II antioxidant enzymes like SOD, CAT, and GSH levels were found to be significantly (P < 0.001) decreased as compared with group I as shown in [Table 2]. There were significant increase (P < 0.05) in SOD, CAT, and GSH levels in groups III, IV, and V when compared with group II. Also there was a significant increase (P < 0.05) in serum SOD, CAT, and GSH levels in group VI when compared with group II.
Effect of E. ganitrus on Kidney Histology
Histological studies revealed that GM-induced renal tubular damage leading to tubular necrosis and pretreatment with E. ganitrus partially prevented damage of renal tubule and glomeruli [Figure 1].
|Figure 1: Histology of kidney after 6-days study period. H and E, ×20 of kidney sections in fi ve different groups of animals revealing signifi cantly higher degree of brush border in renal tubules (red arrowheads) and glomerulus (red asterisk). Cast formation (yellow asterisk), tubular necrosis (yellow arrow heads), and cell detachment (orange arrows) have seen in GM-treated group. Further damage in all treated groups compared with group II shows remarkable improvement after E. ganitrus and MP treatment. H and E = hematoxylin and eosin. a = Group I(CMC), b = Group II(GM 100 mg/kg), c = Group III (GM+EM 100 mg/kg), d = Group IV (GM+EG 200 mg/kg), e=Group V (GM+EM 400 mg/kg), f = Group VI (GM+MP 12.5 mg/kg)|
Click here to view
Effect of E. ganitrus on phagocytic index and neutrophil adhesion assay
In group II the phagocytic index and neutrophil adhesion were found to be significantly (P < 0.001) decreased as compared with group I as shown in [Figure 2]. In groups III, IV, V, and VI there was a significant (P < 0.001) increase in phagocytic index and neutrophil adhesion when compared with group II. Groups III, IV, and V showed a dose-dependent activity on phagocytic index and neutrophil adhesion, respectively, with (P < 0.05) significance when compared with group I [Figure 2].
|Figure 2: Phagocytic index and neutrophil adhesion assay of E. ganitrus. Values are expressed as mean ± SEM of six animals. Superscript letters represent the statistical signifi cance done by ANOVA, followed by Newman-Keuls multiple comparison tests. (a) P < 0.001 indicates comparison made with groups II, III, and VI; (b) P < 0.001 indicates comparison made with groups III, IV, V, and VI; (c) P < 0.05 indicates comparison made with groups IV, V, and VI; (d) P < 0.05 indicates comparison made with group V and group VI; (e) P < 0.001 indicates comparison made with group VI; and (f) P < 0.001 indicates comparison made with group II. SEM = standard error of mean, ANOVA = analysis of variance|
Click here to view
| » Discussion|| |
Nephrotoxicity induced by GM is characterized by an increase in serum creatinine, BUN, uric acid, and albuminuria with severe proximal renal tubular necrosis, followed by deterioration and renal failure. , Serum creatinine concentration is a more potent indicator than the urea concentration in the first phases of kidney disease. Furthermore, urea concentration begins to increase only after parenchymal tissue injury.  In this study serum creatinine, BUN, uric acid, and urine albumin levels were significantly higher in the GM-treated animals, which is an indicator of nephrotoxicity. Coadministration of E. ganitrus in three different doses to nephritic rats resulted significant improvement in the renal function. The mechanism of this protective effect might be due to antioxidant potential of E. ganitrus. These results are in accordance with several other researches, which reported that compounds that have antioxidant properties like resveratrol or garlic extract  partially prevented the increase in the levels of serum creatinine, BUN, uric acid, and albumin in urine against GM-induced nephrotoxicity. In the present investigation, administration of GM resulted in renal injury as a consequence of tubular necrosis and treatment with E. ganitrus extract significantly reduced tubular necrosis and glomeruli damage as shown in [Figure 1].
The toxicity of aminoglycosides, including GM, is related to the generation of ROS in the kidney.  A relationship between nephrotoxicity and oxidative stress has been confirmed in many experimental model. Activities of SOD and CAT enzymes were greatly reduced in GM-treated rats when compared with control group animals. The scavenging of superoxide radicals is achieved through upstream enzyme SOD, which catalyzes the dismutation of superoxide to H 2 O 2. This reaction has a 10,000-fold faster rate than spontaneous dismutation. Reduction in SOD and CAT activities after GM injection has been suggesting that oxidative stress is one of the causes of GM-induced renal damage. The administration of different doses of E. ganitrus in nephritic rats showed increase in SOD and CAT activities may be due the antioxidant property of E. ganitrus. GSH has a very important role in protecting against oxygen free radical damage by providing reducing equivalents for several enzymes. GSH is also a scavenger of hydroxyl radicals and singlet oxygen generation; in the present study, level of GSH in E. ganitrus-treated group is higher when compared with GM-treated animals. The reason for GSH depletion after treatment with E. ganitrus is because of increased GSH consumption in nonenzymatic removal of oxygen radicals. 
The primary function of neutrophils is migration toward the challenge and killing of intracellular microorganisms. Nephrotoxicity leads to reduction in neutrophil function that reflex reduced chemotactic activity.  Hence, in this case the percentage of neutrophils adhered to nylon fiber has been significantly reduced. In treatment groups, there has been increase in adhesion of neutrophils to nylon fiber that reflex its increased chemotactic activity.
Phagocytic index was used to evaluate effect on reticuloendothelial cell-mediated phagocytosis.  Nephrotoxicity leads to increase in macrophages infiltration that results in reduced phagocytic activity. In treatment group, there is increased carbon clearance that reflects increased phagocytic activity of macrophages. Hence E. ganitrus stimulated the reticuloendothelial system by significant increase in the phagocytic index.
| » Conclusion|| |
Present study revealed that administration of E. ganitrus improves renal function in GM-induced nephrotoxicity. Stimulation of antioxidant enzymes along with immunomodulation contributed for restoration of kidney function. Further phytochemical and pharmacological investigations are required to explore the active constituent's and molecular pathways that are responsible for the activity of E. ganitrus. These investigations may have a considerable impact on future drug discovery process for the efficient pharmacotherapeutic management of nephrotoxicity.
| » Acknowledgment|| |
The authors would like to thanks Dr. P. Rajeshwar Reddy, Chairman and the management of Anurag group of Institutions for their support in providing research facilities.
| » References|| |
|1.||Reiter RJ, Tan DX, Sainz RM, Mayo JC, Lopez-Burillo S. Melatonin: Reducing the toxicity and increasing the efficacy of drugs. J Pharm Pharmacol 2002;54:1299-321. |
|2.||Walker PD, Shah SV. Gentamicin enhanced production of hydrogen peroxide by renal cortical mitochondria. Am J Physiol 1987;253:C495-9. |
|3.||Cuzzocrea S, Mazzon E, Dugo L, Serraino I, Di Paola R, Britti D, et al. A role for superoxide in gentamicin-mediated nephropathy in rats. Eur J Pharmacol 2002;450:67-76. |
|4.||Al-Majed AA, Mostafa AM, Al-Rikabi AC, Al-Shabanah OA. Protective effects of oral arabic gum administration on gentamicin -induced nephrotoxicity in rats. Pharmacol Res 2002;46:445-51. |
|5.||Baliga R, Ueda N, Walker PD, Shah SV. Oxidant mechanisms in toxic acute renal failure. Drug Metab Rev 1999;31:971-97. |
|6.||Pedraza-Chaverri L, Maldonado PD, Medina-Campos N, Oliver-Corichi IM, Granados-Silvestre ML, Hernandez-Pando R, et al. Garlic ameliorates gentamicin nephrotoxicity: Relation to antioxidant enzymes. Free Radic Biol Med 2000;29:602-11. |
|7.||Karahan I, Atessahin A, Yilmaz S, Ceribasi AO, Sakin F. Protective effect of lycopene on gentamicin-induced oxidative stress and nephrotoxicity in rats. Toxicology 2005;215:198-204. |
|8.||Karadeniz A, Yildirim A, Simsek N, Kalkan Y, Celebi F. Spirulina platensis protects against gentamicin-induced nephrotoxicity in rats. Phytother Res 2008;22:1506-10. |
|9.||Gagan S, Prabh SS, Mann AS, Shri R. Scientific basis for the chemical constituent and therapeutic use of elacarpus species: A review. Int J Inst Pharm Life Sci 2011;1:267-78. |
|10.||Sarma JK, Bhuyan GC, Koley J, Maity LN, Naikwadi VB. An experimental evaluation of the effect of rudraksha (Elaeocarpus ganitrus roxb) in adrenaline and nicotine induced hypertension. Anc Sci Life 2004;23:1-10. |
|11.||Luo XD, Basile MJ, Kennelly EJ. Polyphenolic antioxidants from the fruits of Chrysophyllum cainito L (Star Apple). J Agric Food Chem 2002;50:1379-82. |
|12.||Singh RK, Acharya SB, Bhattacharya SK. Pharmacological activity of Elaeocarpus sphaericus. Phytother Res 2000;14:36-9. |
|13.||Hule AK, Shah AS, Gambhire MN, Juvekar AR. An evaluation of the antidiabetic effects of Elaeocarpus ganitrus in experimental animals. Indian J Pharmacol 2011;43:56-9. |
|14.||Almeida RN, Navarro DS, Barbosa-Filho JM. Plants with central analgesic activity. Phytomedicine 2001;8:310-22. |
|15.||Singh B, Chopra A, Ishar MP, Sharma A, Raj T. Pharmacognostic and antifungal investigations of Elaeocarpus ganitrus (Rudrakasha). Indian J Pharm Sci 2010;72:261-5. |
|16.||Singh RK, Nath G. Antimicrobial activity of Elaeocarpus sphaericus. Phytother Res 1999;13:448-50. |
|17.||Evans WC. Alkaloids. In: Evans WC, editor. Pharmacognosy. 16 th ed. London: Saunders Elsevier; 1996. p. 353-75. |
|18.||Ghosh MN. Toxicity studies. In: Ghosh MN, editor. Fundamental of Experimental Pharmacology. 4 th ed. Kolkata: Hilton and Company; 1984. p. 176-83. |
|19.||Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem 1951;193:265-75. |
|20.||Marklund S, Marklund G. Involvement of the superoxide anion radical in auto oxidation of pyrogallol and a convenient assay for peroxide dismutase. Eur J Biochem 1974;47:469-74. |
|21.||Goth L. A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta 1991;196:143-51. |
|22.||Wilkonson PC. Neutrophil adhesion test. In: Vane JK, Ferreria SH, editors. Experimental Pharmacology. 1 st ed. Berlin: Springer-Verlag; 1978. p. 109-12. |
|23.||Gonda R, Tomoda M, Shiminzu N, Kanari M. Characterization of an acidic polysaccharide from the seeds of Malva verticillata stimulating the phagocytic activity of cell the RES. Planta Med 1990;56:73-6. |
|24.||Gilbert DN, Wood CA, Kohlepp PW, Houghton DC, Finkbeiner HC, Lindsley J, et al. Polyaspartic acid prevents experimental aminoglycoside nephrotoxicity. J Infect Dis 1989;159:945-53. |
|25.||Hule AK, Juvekar AR. Evaluation of immunomodulatory effects of methanolic extract of Elaeocarpus ganitrus seeds. J Nat Remed 2010;10:1-10. |
[Figure 1], [Figure 2]
[Table 1], [Table 2]