|Year : 2014 | Volume
| Issue : 2 | Page : 166-170
Mentha piperita in nephrotoxicity - a possible intervention to ameliorate renal derangements associated with gentamicin
Naveed Ullah1, Mir Azam Khan2, Taous Khan3, Afzal Haq Asif4, Waqar Ahmad2
1 Department of Pharmacy, University of Swabi, Swabi, Pakistan
2 University of Malakand, Chakdara, Pakistan
3 Comsats Institute of Information Technology, Abbottabad, Pakistan
4 Department of Pharmacology, Frontier Medical College, Abbottabad, Pakistan
|Date of Submission||29-Jan-2013|
|Date of Decision||16-Apr-2013|
|Date of Acceptance||21-Jan-2014|
|Date of Web Publication||24-Mar-2014|
Department of Pharmacy, University of Swabi, Swabi
Source of Support: Higher Education Commission, Government of
Pakistan., Conflict of Interest: None
Objective: Free radical generation has a strong role in the pathogenesis of renal damage associated with the use of gentamicin. Therefore, the present study was carried out to evaluate the renoprotective effect of Mentha piperita against gentamicin induced nephrotoxicity.
Materials and Methods: A total of 24 male rabbits were divided into 4 groups receiving normal saline, gentamicin, M. piperita extract and co-therapy of extract and gentamicin respectively. Gentamicin was provided as 80 mg/kg/day intramuscularly and extract was given 200 mg/kg/day orally for a period of 21 days. Serum and urinary biochemical parameters and histological changes were studied for each group. The impact of the extract on the antibacterial action of gentamicin was also evaluated.
Results: Animals treated with gentamicin showed derangements in serum and urinary biochemical parameters. These alterations were reversed by treatment with M. piperita extract. The histological changes showed in gentamicin group were also reverted by treatment with the extract. Further the plant did not influence the efficacy of gentamicin with respect to its antimicrobial properties.
Conclusion: Co-therapy of M. piperita with gentamicin successfully attenuated biochemical kidney functioning derangements and morphological changes associated with gentamicin.
Keywords: Aminoglycosides, gentamicin, Mentha piperita, nephroprotective, peppermint
|How to cite this article:|
Ullah N, Khan MA, Khan T, Asif AH, Ahmad W. Mentha piperita in nephrotoxicity - a possible intervention to ameliorate renal derangements associated with gentamicin. Indian J Pharmacol 2014;46:166-70
|How to cite this URL:|
Ullah N, Khan MA, Khan T, Asif AH, Ahmad W. Mentha piperita in nephrotoxicity - a possible intervention to ameliorate renal derangements associated with gentamicin. Indian J Pharmacol [serial online] 2014 [cited 2019 Sep 20];46:166-70. Available from: http://www.ijp-online.com/text.asp?2014/46/2/166/129309
| » Introduction|| |
Mentha piperita L. belongs to family Lamiaceae, commonly known as mint, spearmint and peppermint. The plant is widely used as an anti-spasmodic, carminative, anti-motion sickness and anthelmintic.  Brazilians have used the plant for the treatment of nausea, vomiting, common cold, fever, bronchitis and for stimulation of appetite.  The plant has also been reported to have antimicrobial properties. , Decoction of M. piperita leaves have been used as tonic, digestive and anti-inflammatory in Iranian Traditional Medicine.  The plant extract has been shown to contain strong flavonoid and phenolic compounds, which might responsible for their concentration dependent free radical scavenging properties. ,,
Gentamicin, an aminoglycoside, is known for its nephrotoxicity and one of the possible mechanisms suggested is damage due to generation of free radicals.  Due to the wide use of plant extract for the treatment of different ailments and especially due to their concentration dependent free radical scavenging properties, it was thought worthwhile to explore the protective effect of M. piperita against gentamicin-induced renal damage.
| » Materials and Methods|| |
A total of 24 male rabbits of mixed breed (1-1.5 kg) were kept in the animal house of Frontier Medical College, Abbottabad, 15 days before the start of experiment for acclimatization after the approval of the project by the University Animal Ethics Committee. Animal handling and care were strictly performed according to the guidelines of the university (University of Malakand) along with international laws and policies (National Institutes of Health Guide for the Care and Use of Laboratory Animals, NIH Publication no. 85-23, 1985). All the animals were maintained on same diet at 12 h light/dark cycle. Injection gentamicin 80 mg/ml (Merck Private Ltd.) was purchased from local market, Abbottabad, Pakistan.
Plant Material and Extraction
Fresh plant leaves of M. piperita was collected from Abbottabad after identification by Professor Umar Farooq, Department of Botany, G.P.G College Abbottabad and the voucher specimen (1028) was submitted to the herbarium of the same college. Extraction was done with ethanol after drying the leaves under shade. The ethanol was than evaporated by rotary evaporator (r210, Germany).
were divided into four groups of 6 each and each group was treated independently as follows:
- Group C received 0.9% saline only 2 ml/kg (i.m) for 21 days
- Group G received gentamicin 80 mg/kg (i.m) for 21 days
- Group GM-pi received gentamicin 80 mg/kg (i.m) + M. piperita 200 mg/kg (p.o) for 21 days
- Group M-pi received M. piperita 200 mg/kg (p.o) for 21 days.
Blood from the marginal vessel of the pinna and urine samples were collected on day 0, 11 th and 21 st of experimental period for measurement of blood urea nitrogen, serum creatinine, creatinine clearance, serum uric acid, serum electrolytes, urinary volume, urinary creatinine, urinary proteins, urinary alkaline phosphatase and urinary lactate dehydrogenase to find out the protective role of plant extract against gentamicin induced nephrotoxicity.
Bertholot's indophenol assay was used for the estimation of blood urea nitrogen while serum and urinary creatinine was measured by Jaffe reaction method. Serum uric acid was measured by using commercially available reagents with the help of chemistry analyzer (Merck, Germany).  Flame photometric method was used for estimation of serum sodium and serum potassium while serum calcium was estimated by cresolphthalein complex one method.  Alkaline phosphatase and lactate dehydrogenase was measured by using commercially available reagents (Randox laboratories, UK), following German Society of Clinical Chemistry. 
Three rabbits in each group were sacrificed on day 21 of study period for examination of the kidneys. Each kidney was isolated and fixed in 10% formalin. Ethanol was applied in ascending order followed by two grades of absolute alcohol and xylene. Tissues were fixed with the help of wax. Blocks were cut down by using rotatory microtome (Micros, Germany). Hematoxylin and Eosin dyes were used for the staining of slides and examined under microscope (Germany).
Impact of Plant Extract on Antibacterial Activity of Gentamicin
Effect of the plant extract on antibacterial activity of gentamicin was tested against five bacterial strains including Gram-negative bacteria, i.e. Salmonella typhi, Escherichia coli, Pseudomonas pickettii and Proteus mirabilis and Gram-positive bacteria, i.e. Micrococcus luteus. Bacterial cultures were provided by Ayub Medical College, Abbottabad. Agar well diffusion method was followed.  The plates were sterilized in autoclave (Hirayama) and prepared in aseptic condition under laminar flow hood. The bacteria were cultured and incubated for 24 h at 37°C. Few colonies from these cultures were inoculated on Mueller-Hinton Broth and the inoculum was spread over the plates. The seeded plates were then allowed to dry for 20 min in the incubator (Mammert, Germany). Uniform sized wells were cut down on the surface of Mueller-Hinton Agar with the help of a standard cork borer. The plant extract at a concentration of 500 mg/ml and gentamicin at a concentration of 40 mg/ml were used in combination. The inoculated plates were then incubated for 24 h at 37°C and the zone of inhibition was measured to the nearest millimeter with the help of colony counter (Sunte ×570, Taiwan).
All experimental groups were compared by one-way analysis of variance (ANOVA) followed by Dunnett test and the results were expressed as mean ± standard error of mean the difference between groups was considered to be statistically significance if the P < 0.05.
| » Results|| |
Significant increase in blood urea nitrogen, serum creatinine and serum uric acid levels were observed in Group G, on day 11 and 21 when compared with control group (P < 0.0001). Group GM-pi and M-pi showed a significant decrease in these parameters when compared with Group G (P < 0.0001) on day 11 and 21 [Table 1]. Significant decrease in creatinine clearance was observed in Group G on day 11 and 21 when compared with Group C (P < 0.001). Group GM-pi and M-pi showed a significant increase in creatinine clearance on day 11 and 21 when compared with Group G (P < 0.0001) [Table 1].
|Table 1: Effect of ethanolic extract of M. piperita on serum biochemical parameters in gentamicin induced nephrotoxicity|
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Serum sodium was unchanged throughout the study period in all treated groups. Significant decrease in serum potassium and calcium in Group G was seen on day 11 and 21 with respect to Group C (P < 0.0001). Significant increase in serum potassium was observed in Groups GM-pi and M-pi on day 21 while significant increase in serum calcium levels was observed on both day 11 and 21 when compared with Group G (P < 0.0001) [Table 1].
Urinary volume was observed to be statistically unchanged on day 11 th in all experimental groups, while on day 21 the urinary volume of Group G was significantly decreased when compared with Group C (P < 0.001). Group M-pi on day 21 had a significantly higher urine volume when compared with Group G (P < 0.0001). Microscopic examination of urine revealed the presence of significant amount of red blood cells, leukocytes and renal casts in Group G animals. These findings were not seen in Groups C, GM-pi and M-pi.
Urinary excretion of protein and lactate dehydrogenase was significantly increased in Group G on day 11 and 21 as compared to Group C (P < 0.05 and 0.0001 respectively). Group GM-pi and M-pi were found to have significantly decreased urinary protein levels as compared to Group G on day 21; however this difference was not significant when measured on day 11 th of study [Table 2]. Significant decrease in urinary lactate dehydrogenase levels was noted in Group GM-pi and M-pi on day 11 with respect to Group G (P < 0.0001) [Table 2]. Urinary excretion of alkaline phosphatase was noted unchanged in all experimental groups throughout the study period.
|Table 2: Effect of ethanolic extract of M. piperita on urinary biochemical parameters in gentamicin induced nephrotoxicity|
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Histological examination of the kidneys of Group G showed proximal tubular necrosis with loss of cellular pattern. Glomerular atrophy and ruptured tubules with hydropic changes were also observed [Figure 1]b. While in case of Group C animals' normal tubules with no evidence of necrosis and normal glomeruli or hydropic changes were observed [Figure 1]a. Groups GM-pi and M-pi also showed normal histology with no common abnormality or significant toxicity [Figure 1]c and d.
|Figure 1: Photomicrograph showing histopatological examination of renal cortex (a) Group C, presenting no common abnormality (b) Group G, presenting glomerular atropy with hydropic changes and proximal tubular cell necrosis with loss of cellular pattern (c) Group GM-pi, presenting normal glomeruli with no evidence of necrosis or ruptured tubules (d) Group M-pi, also presenting normal structures|
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Impact of Plant Extract on Antibacterial Activity of Gentamicin
The zone of inhibition measured for gentamicin alone and in combination with plant extract against S. typhi was 31 mm. In the other Gram-negative bacterial cultures, the zone of inhibition of gentamicin in combination with M. piperita extract was greater than with gentamicin alone (E. coli - 29 mm vs. 20 mm, P. pickettii - 42 mm vs. 34 mm, P. mirabilis - 18 mm vs. 15 mm). The zone of inhibition measured for gentamicin against M. luteus was 34 mm while for the combination was 42 mm.
| » Discussion|| |
The current study was aimed to explore the nephroprotective effects of M. piperita against gentamicin-induced renal damage. Different doses of gentamicin, ranging between 40 and 100 mg/kg/day have been used by different researchers to produce significant nephrotoxictiy  and as it has been reported that the nephrotoxic effects of gentamicin can be produced only when the drug is used at 5-10 times the normal therapeutic dose. , Therefore in the current study, daily dose of 80 mg/kg of gentamicin was selected to produce strong nephrotoxic effect.
Increase in serum creatinine with the rise in blood urea nitrogen and a significant fall in creatinine clearance has been reported previously with gentamicin. ,, In the present study, significant rise in the serum creatinine, blood urea nitrogen and serum uric acid with fall in creatinine clearance were observed in Group G animals when compared with control, which was reversed to almost control values in the extract treated animals. This showed the protective role of M. piperita against toxic effects of gentamicin on kidney.
Gentamicin associated rise in urinary excretion of sodium and potassium has been reported previously  and this hypokalemia is mostly due to the chronic administration of the drug. Similarly, in the current study significant hypokalemia was observed in Group G animals, different from Group C and all other treated groups. , Like control group animals, the animals treated with plant extract alone and in combination with gentamicin did not produce fall in serum potassium.  In agreement with the previous reports, fall in serum calcium was observed in Group G animals, which was significantly different from Group C, Group GM-pi and Group M-pi. , Further, it has also observed in other studies that large doses of gentamicin altered serum sodium level, however in the present study no significant alteration in serum sodium level was observed. 
Rise in serum creatinine depends upon the extent of tubular necrosis  which may due to the blocking of tubules by the necrotic debris as observed in renal histopathological sections in the current study. Significant rise of serum creatinine was observed in Group G animals with proximal tubular necrosis. However, it has been reported that alteration in the renal functioning parameters and proximal tubular necrosis are independent. ,
Urinary volume decreased in Group G animals with an increase in urinary protein excretion which was significantly different from control group animals. However, urinary volume and urinary protein excretion in animals treated with co-therapy of gentamicin and M. piperita and M. piperita alone was similar with Group G animals on day 11 th , which further changed on day 21 st of study period. Urinary alkaline phosphatase and lactate dehydrogenase was measured to explore the nephro-protective effects of plant extract as used previously.  Urinary alkaline phosphatase excretion remained unchanged throughout the study period. However, urinary lactate dehydrogenase significantly increased in Group G animals on day 11 th of study period. Further, animals treated with co-therapy of gentamicin and M. piperita and M. piperita alone were significantly different from Group G animals on day 11 th but similar on day 21 st of study period showed their importance in early detection of nephrotoxicity.
Histological examination of kidney revealed the presence of proximal tubular necrosis, regenerating cells and hydropic changes in Group G animals which were in agreement with previous reports. , Significant increase in urinary protein excretion may because of the presence of hyaline and renal casts in the tubules which may lead to cellular degeneration. Renal casts may block the tubules and lead to renal damage. , Cellular derangements, hydropic changes and the presence of necrosis and cast cells were not significantly detected in control group animals, extract treated and animals treated with co-therapy of M. piperita and gentamicin.
The plant extract was also screened for its impact on the antibacterial activity of gentamicin. M. piperita has been previously reported to have strong antibacterial properties.  Similarly, in the current study, the plant extract increased the zone of inhibition of gentamicin against several bacteria.
It can be concluded that concurrent administration M. piperita successfully prevented renal damage associated with gentamicin, explored by various biochemical and histological examinations. Alteration in mean body weight, blood urea nitrogen, creatinine, creatinine clearance, uric acid, urinary protein and enzymes excretion associated with gentamicin were reduced by treating animals simultaneously with crude extract of M. piperita. Further, the study also shows that concomitant use of M. piperita does not decline the efficacy of gentamicin with respect to its antibacterial activities. Further studies need to be undertaken in order to confirm these findings and its extrapolation in humans.
| » Acknowledgments|| |
This study was financially supported by HEC, Government of Pakistan through indigenous PhD scholarship.
| » References|| |
|1.||Lorenzi H, Matos FJ. Plantas Medicinais no Brasil, Nativas e Exóticas. Nova Odessa: Instituto Plantarum; 2008. |
|2.||Akdogan M, Kilinç I, Oncu M, Karaoz E, Delibas N. Investigation of biochemical and histopathological effects of Mentha piperita L. and Mentha spicata L. on kidney tissue in rats. Hum Exp Toxicol 2003;22:213-9. |
|3.||Romero-Jiménez M, Campos-Sánchez J, Analla M, Muñoz-Serrano A, Alonso-Moraga A. Genotoxicity and anti-genotoxicity of some traditional medicinal herbs. Mutat Res 2005;585:147-55. |
|4.||Mimica-Dukiæ N, Bozin B, Sokoviæ M, Mihajloviæ B, Matavulj M. Antimicrobial and antioxidant activities of three Mentha species essential oils. Planta Med 2003;69:413-9. |
|5.||Zargari A. Medicinal Plants. Tehran: Tehran University Publications; 1990. p. 4-20. |
|6.||Pramila DM, Xavier RI, Marimuthu KI, Kathiresan SI, Khoo ML, Senthilkumar MS, et al. Phytochemical analysis and antimicrobial potential of methanolic leaf extract of peppermint (M. piperita: Lamiaceae). J Med Plant Res 2012;6:331-5. |
|7.||Dorman HJ, Koºar M, Baºer KH, Hiltunen R. Phenolic profile and antioxidant evaluation of Mentha xpiperita L.(peppermint) extracts. Nat Prod Commun 2009;4:535-42. |
|8.||Samarth RM, Samarth M. Protection against radiation-induced testicular damage in Swiss albino mice by Mentha piperita (Linn.). Basic Clin Pharmacol Toxicol 2009;104:329-34. |
|9.||Ali BH, Mousa HM. Effect of dimethyl sulfoxide on gentamicin-induced nephrotoxicity in rats. Hum Exp Toxicol 2001;20:199-203. |
|10.||Smith ST. Non protein nitrogen. In: Bishop ML, Duben-Von JH, Fody EP, editors. Clinical Chemistry, Principles, Procedures, Correlations. Philadelphia: JB Lippincott Company; 1985. p. 411-23. |
|11.||Blosser N. Electrolytes. In: Bishop ML, Duben-VonLaufen JL, Fody EP, editors. Clinical Chemistry, Principles, procedures, correlations. Philadelphia: JB Lippincott Company; 1985. p. 263-89. |
|12.||Johnson AM, Rohlfs EM, Silverman LM. Proteins. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. 3 rd ed. Philadelphia: WB Saunders; 1999. p. 477-540. |
|13.||Tortora GJ, Funke BR, Case CL. Microbiology, an Introduction. 7 th ed. San Francisco: Benjamin Cummings; 2005. p. 533. |
|14.||Houghton DC, Lee D, Gilbert DN, Bennett WM. Chronic gentamicin nephrotoxicity. Continued tubular injury with preserved glomerular filtration function. Am J Pathol 1986;123:183-94. |
|15.||Bennett WM, Elzinger LW, Porter GA. Tubulointerstitial disease and toxic nephropathy. In: Brenner BM, Rector JR, editors. The Kidney. Philadelphia: WB Saunders Company; 1991. p. 1451-95. |
|16.||Tulkens PM. Nephrotoxicity of aminoglycoside antibiotics. Toxicol Lett 1989;46:107-23. |
|17.||Bennett WM, Plamp CE 3 rd , Parker RA, Gilbert DN, Houghton DC, Porter GA. Alterations in organic ion transport induced by gentamicin nephrotoxicity in the rat. J Lab Clin Med 1980;95:32-9. |
|18.||Moghaddam AH, Javaheri M, Nabavi SF, Mahdavi MR, Nabavi SM, Ebrahimzadeh MA. Protective role of Pleurotus porrigens (Angel's wings) against gentamicin-induced nephrotoxicty in mice. Eur Rev Med Pharmacol Sci 2010;14:1011-4. |
|19.||Derakhshanfar A, Bidadkosh A, Kazeminia S. Vitamin E protection against gentamicin induced nephrotoxicity in rats: a biochemical and histopathologic study. Iran J Vet Res 2007;8:231-8. |
|20.||Cronin RE, Bulger RE, Southern P, Henrich WL. Natural history of aminoglycoside nephrotoxicity in the dog. J Lab Clin Med 1980;95:463-74. |
|21.||Asif AH, Rasool ST, Khan TM. Pyridoxal phosphate a possible intervention to prevent aminoglycoside induced electrolyte imbalance. J Clin Res Bioeth 2012;3:124. |
|22.||Lambie AT. Disturbances in water, electrolyte and acid-base balance. In: Edward CR, Bouchieer IA, editors. Principles and Practice of Medicine. 16 th ed. Edinburgh: Churchill Livingstone; 1991. p. 203-28. |
|23.||Solez K. Pathogenesis of acute renal failure. In: International Review of Experimental Pathology. New York: Academic Press; 1983. p. 321-6. |
|24.||Luft FC, Yum MN, Kleit SA. The effect of concomitant mercuric chloride and gentamicin on kidney function and structure in the rat. J Lab Clin Med 1977;89:622-31. |
|25.||McCracken GH. Aminoglycoside toxicity in infants and children. Am J Med 1986;30:171-8. |
[Table 1], [Table 2]