| Abstract|| |
Matricaria chamomilla is extensively consumed as a tea or tonic. Despite its widespread use as a home remedy, relatively few trials evaluated its benefits in nephro protection. Hence, this study evaluates the protective role of M. chamomilla in cisplatin nephrotoxicity rat model. The study was conducted on 32 rats divided into four groups. The first group (G1) was injected with saline intra-peritoneally (IP); G2 was injected with 5 mg/kg cisplatin on day 0 of the experiment and repeated four times, with five days free interval. G3 and G4 were injected daily with M. chamomilla (50 mg/kg) IP, starting five days before the experiment (-5 day); in addition, G4 was injected with cisplatin. On day 16, animals were scarified and serum and/or kidney tissue was used to determine: (a) kidney function tests (serum urea, creatinine, gamma glutamyl transferase (GGT), NAG, β-gal), (b) oxidative stress indices (NO, LPO), (c) antioxidant activities (SOD, GSH, total thiols), (d) apoptotic indices (Cathepsin D, DNA fragmentation) and (e) mineral (calcium). M. chamomilla significantly increased the body weight, normalized the kidney functions, improved the apoptotic markers, reduced the oxidative stress markers and corrected the hypo-calcemia that resulted from cisplatin nephrotoxicity. M. chamomilla is a promising nephro-protective compound reducing cisplatin nephrotoxicity most probably by its antioxidant activities and inhibition of gamma glutamyl transferase activity.
|How to cite this article:|
Salama RH. Matricaria chamomilla attenuates cisplatin nephrotoxicity. Saudi J Kidney Dis Transpl 2012;23:765-72
| Introduction|| |
Cisplatin is a major antineoplastic drug for the treatment of solid tumors, but it has dose-dependent renal toxicity. It has multiple intra-cellular effects, causing direct cytotoxicity with reactive oxygen species, activating mitogen-activated protein kinases, inducing apoptosis and stimulating inflammation and fibrogenesis.  Chamomile (Matricaria recutita L., Chamomilla recutita L., Matricaria chamomilla) is one of the most popular single-ingredient herbal tea, or tisanes. Chamomile tea, brewed from dried flower heads, has been used traditionally for medicinal purposes. Evidence-based information regarding the bioactivity of this herb is presented. Chamomile has moderate antioxidant and antimicrobial activities and significant anti-platelet activity in vitro. Animal model studies indicate potent anti-inflammatory action, some antimutagenic and cholesterol-lowering activities as well as antispasmotic and anxiolytic effects.  Its methanolic extract showed potent neuroprotective activity against global cerebral ischemia/reperfusion injury-induced oxidative stress in rats.  There are numerous varieties of Chamomile, but the two most popular are Roman chamomile and German chamomile. German chamomile is called Matricaria chamomilla, and is considered the more potent of the two, and has received more scientific evaluation.  Evidence recommend German chamomile for short-term relief of mild insomnia in cancer patients.  This study aimed to evaluate the protective effects of M. chamomilla in cisplatin nephrotoxicity rat model.
| Materials and Methods|| |
Preparation of Matricaria chamomilla and cisplatin
M. chamomilla (dry leaves and flowers) was selected with a fair degree of quality assurance from the Faculty of Pharmacy, Pharmacognosy Department, Assiut University, Egypt. The identity of the plant was verified by the Center of Medicinal, Aromatic and Poisonous Plants and a voucher specimen was kept on record in the herbarium of the Faculty of Pharmacy. The dry leaves were crushed to powder and 25 g of the powder was extracted with 500 mL 95% ethanol overnight with continuous stirring. This was repeated for three successive days. The pooled extracts (1500 mL) were evaporated to render the product alcohol-free.  One gram of the product was reconstituted with saline until the final concentration was 50 mg/mL and stored at 4C ready for use.
Cisplatin was obtained from Bristol-Myers Squibb Company, Princeton, New Jersey, USA. A vial of 50 mg was reconstituted with 25 mL saline immediately before injection, with a final concentration of 2 mg/mL.
The study was carried out on 32 healthy adult male Sprague-Dawley rats weighing 200-300 g. Their ages ranged from 22 to 24 weeks. The animals were housed conventionally in clean cages and fed with standard food and water ad libitum. The animals were housed in groups in 12-h light/dark cycles. The care and treatment of the animals were approved and performed according to the guidelines of the Animal House of Faculty of Medicine, Assiut University, Egypt. The rats were divided randomly into four groups of eight animals each. The first group (G1) was the healthy reference (HR) group, injected intraperitoneally (IP) with 1 mL saline. The second group (G2) was injected IP with 5 mg/kg cisplatin on day 0 of the experiment and repeated four times, with five days free interval in between. By the 5 th day, up to 43% of the administered cisplatin was recovered in the urine.  G3 and G4 were injected IP with M. chamomilla (50 mg/kg). The injection started five days before the experiment (-5 day) and continued daily till the end of the experiment. In addition, G4 received cisplatin injection 1 h after M. chamomilla in the same manner as group 2. On day 16, the animals were weighed, blood samples obtained by preoribital puncture, then sacrificed and both kidneys were removed and weighed. Kidneys were homogenized in 6 mL ice cold saline, then centrifuged at 4000 x g for 10 min at 4C and the supernatant was conserved at -70C for biochemical assays.
- Serum samples were used for determi nation of
1.1. Kidney function tests: serum urea, creatinine determined by using kit from Stanbio, Boerne, TX, USA, and gamma glutamyl transferase (GGT) by using kit from QCA, Amposta (Tarragona), Spain.
1.2. Oxidative stress indices: nitric oxide (NO) measured as total nitrite  and lipid peroxidation (LPO) as thio barbituric acid-reactive substances. 
1.3. Antioxidants: Superoxide dismutase activity (SOD)10 and total thiols measured by chemical method. 
1.4. Serum level of Ca+2 determined by an atomic absorption spectrophotometer (Buck, E. Norwalk Scientific, USA; model 210 VGP) using nitrous oxide/ acetone flame absorption at wave length 422.7 nm.
- Supernatants of kidney homogenates were used for determination of:
2.1. Proximal convoluted tubules function tests by beta-N-acetylglucosaminidase (NAG) and β-gGalactosidase (β-Gal). 
2.2. Oxidative stress index: LPO was determined as thiobarbituric acid reactive substances. 
2.3. Antioxidants: SOD  and reduced glutathione (GSH) determined by a spectrophotometer. 
2.4. Apoptotic indices: Cathepsin D was determined by chemical methods, as hemoglobin splitting activity of cathepsin  and DNA fragmentation by colorimetric method using diphenylamine (98% v/v glacial acetic acid; 1.5% v/v sulfuric acid; and 0.5% v/v of 1.6% acetaldehyde).  2.5. Total protein was measured using a kit from Stanbio USA.
| Statistical Analysis|| |
Data were analyzed using SPSS version 16. Values were expressed as mean SE. The difference between the HR group (G1) and the cisplatin group (G2) or between groups that received M. chamomilla only (G3) or in combination with cisplatin group (G4) for each parameter was assessed by the Mann-Whitney U test. We performed one way analysis of variance (ANOVA) followed by post hoc test (Tukey HSD) for multiple comparison between cisplatin group (G2) and all groups. The correlation coefficient was calculated to assess the relation between the parameters. The results were considered statistically significant at P≤0.05.
| Results|| |
The clinical data of animals are shown in [Figure 1] (A-C). The increase of body weight in the HR group was 3.99% and in the G3 group, which received M. chamomilla only, was 3.38%. On the other hand, there was a reduction in body weight in G2, which received cisplatin only, by 7.59% and in G4, which received cisplatin and M. chamomilla, by 3.31%.
|Figure 1: A and B: Changes in the body and kidney weights in the different treated groups of animals.|
Figure 1: C. Percentage of kidney weight to body weight (after treatment).
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Cisplatin caused elevation in all kidney function indices. Co-administration of M. chamomilla with cisplatin attenuated the toxic effects of cisplatin in kidney by decreasing the levels of urea, creatinine, NAG, β-GAL and GGT [Table 1]. There was a correlation (r = 0.731, P < 0.05) between GGT and NAG under the effect of cisplatin (G2). Cisplatin caused significance elevation in NO and LPO and a reduction in SOD, total thiols and GSH, and an increase in cathepsin D and DNA fragmentation [Table 2], [Table 3] and [Table 4] compared with controls (G1).
|Table 1: Kidney function test in rats treated with cisplatin only (G2) or in combination with M. chamomilla (G4) compared with controls (G1) who received saline (G3) and those who received M. chamomilla.|
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|Table 2: Oxidative stress in rats treated with cisplatin only (G2) or in combination with M. chamomilla (G4) compared with controls (G1) received saline, or (G3) received M. charmomilla.|
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|Table 3: Antioxidants in rats treated with cisplatin only (G2) or in combination with M. chamomilla (G4) compared with controls (G1) who received saline or (G3) those who received M. chamomilla.|
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|Table 4: Apoptotic markers in rats treated with cisplatin only (G2) or in combination with M. chamomilla (G4) compared with controls (G1) who received saline or (G3) and those who received M. chamomilla.|
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Co-administration of M. chamomilla with cisplatin provided better protection from oxidative stress and apoptosis, as shown in [Table 2] and [Table 3]. Cisplatin caused a significant decrease in serum Ca 2 + . However, co-administration of M. chamomilla with cisplatin (G4) provided better protection from cisplatin-induced hypocalcemia [Figure 2].
|Figure 2: Serum levels of calcium in rats treated with cisplatin only (G2) or in combination with M. chamomilla (G4) compared with controls (G1) that received saline or (G3) those who received M. chamomilla.|
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| Discussion|| |
M. chamomilla is a well-known medicinal plant used as carminative, analgesic and anticonvulsant in traditional medicine.  The extract of M. chamomilla possesses creditable effects on seizure induced by picrotoxin.  Bisabololoxide A is the principle constituent of some bio-activities of German chamomile, including anti-inflammatory, gastrointestinal and anti-pruritic actions. 
In this study (cisplatin 5 mg/kg, four doses, time of experiment 21 days), there was a 7.59% decrease in body weight in the cisplatin-treated group compared with 14% decrease in body weights in another study (cisplatin 15 mg/kg, time of experiment five days).  In this study, there was a 1.4-times increase of kidney weight to body weight after cisplatin treatment. This could be explained by the marked decrease in the total body weight in the cisplatin-treated group. In other studies, there were 1.7- and two-times increase, respectively, in kidney weight to body weight in the cisplatin-treated animals. , The co-administration of M. chamomilla with cisplatin reduced this ratio when compared with the cisplatin treated group; this may be due to relatively less reduction in the total body weight in this group (G4).
According to the cut-off values for the normal range of blood urea nitrogen (≤40 mg/dL) and serum creatinine values (≤0.2 mg/dL) based on the values obtained from normal untreated mice  ; our results disclosed 2.4- and 1.7-times increase of these values, respectively, in the cisplatin-treated group when compared with the control group. In other studies, the increase in the urea levels was 3.7- and two-times and the creatinine levels was 2.3- and 4.5-times. , Furthermore, the cisplatin group in our study showed significant elevation of NAG and β-GAL levels when compared with the control group. This elevation in NAG levels is usually due to early impaired renal function and renal tubular damage.  These changes were minimized in the group that received cisplatin with M. chamomilla (G4). During cisplatin chemotherapy, the oxidative stress associated with increased generation on reactive oxygen metabolites (ROM) can cause LPO in the kidneys and decreased levels of antioxidants and antioxidant enzymes.  M. chamomilla methanolic extracts revealed a dose-dependent nephro-protective activity by significant decrease in LPO and increase in the SOD, catalase (CAT), glutathione (GSH) and total thiol levels in the chamomilla extract-treated groups compared with the ischemia/re-perfusion group.  In this study, oxidative stress induced by cisplatin manifested as an elevation in nitric oxide (NO) and lipid peroxide levels and reduction of antioxidants, SOD, GSH and total thiol levels [Table 2] and [Table 3]. Increased levels of LPO in serum and tissue homogenates in cisplatin-treated animals was reported. , In contrast, a decrease in NO levels in cisplatin-treated animals when compared with controls was detected.  Moreover, there was a decrease in the antioxidant, such as GSH and SOD, activities. 
The expression of cathepsin D, the lysosomal protease, had been shown to increase with protein degradation that occurred during apoptosis.  In this study, cisplatin caused significant increase in cathepsin D levels and DNA fragmentation. Animals that received cisplatin with M. chamomilla revealed a significant reduction in these parameters.
GGT, a key enzyme of GSH metabolism, can modulate crucial redox-sensitive functions, such as antioxidant/antitoxic defenses and cellular proliferative/apoptotic balance.  The highest level of activity was on the luminal surface of the proximal tubule cells in the kidney. Acivicin, an inhibitor of GGT, blocks nephrotoxicity of cisplatin in rats.  GGT cleaves the gamma-glutamyl group of the GSH-conjugate and aminopeptidase cleaves the cysteinyl-glycine bond and results in a platinum-cys-teine conjugate. Finally, the cysteine conjugate is metabolized by cysteine-s-conjugate beta-lyase to reactive thiol.  In this study, M. chamomilla decreased the levels of GGT significantly. Moreover, M. chamomilla did not contain glutamic or methionine to support the regeneration of GSH.  However, chamazulene, a constituent of M. chamomilla, affects free radical processes and inhibits LPO in a concentration- and time-dependent manner. 
Calcium is the most important mineral that is affected by cisplatinum administration. , Calcium released from intracellular calcium storage in the early phase of nephrotoxicity causes oxidative stress in the renal tubular epithelial cells.  In this study, co-administration of M. chamomilla with cisplatin caused a significant improvement in cisplatin-induced hypocalcemia.
The minimal side-effects of M. chamomilla, as it has been used as a tea for a long time, and good protection of kidney may encourage physicians to consider it as an adjuvant with chemotherapy, and human studies accordingly are warranted.
| References|| |
|1.||Yao X, Panichpisal K, Kurtzman N, Nugent K. Cisplatin nephrotoxicity: a review. Am J Med Sci 2007;334:115-24. |
|2.||McKay DL, Blumberg JB. A review of the bioactivity and potential health benefits of chamomile tea (Matricaria recutita L.). Phytother Res 2006;20:519-30. |
|3.||Chandrashekhar VM, Ranpariya VL, Parashar A, Muchandi AA, Ganapaty S. Neuroprotective activity of Matricaria recutita Linn against global model of ischemia in rats. J Ethno-pharmacol 2010;127:645-51. |
|4.||Ali BH, Blunden G. Pharmacological and toxicological properties of Nigella sativa. Phytother Res 2003;17:299-305. |
|5.||Block KI, Gyllenhaal C, Mead MN. Safety and efficacy of herbal sedatives in cancer care. Integr Cancer Ther 2004;3:128-48. |
|6.||El-Daly E. Protective effect of cysteine and vitamin E, Crocus sativus and Nigella sativa extracts on cisplatin-induced toxicity in rats. J Pharm Belg 1998;53:87-95. |
|7.||Lipp H, Hartmann J. Platinum compounds: metabolism, toxicity and supportive strategies. Schweiz. Rundsch Med Prax 2005;94:187-98. |
|8.||Van Bezoijen R, Ederveen A, Kloosterboer H, Papapoulos S, Lowik C. Plasma nitrate and nitrite level are regulated by ovarian steroids but do not correlate with trabecular bone mineral density in rats. J Endocrinology 1998; 159:27-34. |
|9.||Thayer W. Lipid peroxide in rats treated chronically with adriamycin. Biochem Pharmacol 1984;33:2259-63. |
|10.||Misra H, Fridovich I. The role of super oxide ion in the oxidation of the epinephrine and a simple assay for superoxide dismutase. J Biol Che 1972;247:3170-87. |
|11.||Ellman M. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7. |
|12.||Maruhn D. rapid colorimetric assay by â-Galactosidase and N- acetyl-â glucosaminidase in human urine. Clin Chim Acta 1976;73:453-61. |
|13.||Beutler E, Duron O, Kelly B. Improved method for determination of blood glutathione. J Lab Clin Med 1963;61:882-8. |
|14.||Kühn S, deKock M. A preliminary study of elevated alkaline phosphatase and cathepsin in bronchial aspirates of patients with lung cancer and bronchitis. Chest 1975;38:326-30. |
|15.||Paradones C, Illera V, Peckham D, Stunz L. and Ashman R. Regulation of apoptosis in vitro in mature spleen T cells. J Immun 1993; 151:3521-9. |
|16.||Bonevski B, Wilson A, Henry DA. An analysis of news media coverage of complementary and alternative medicine. PLoS ONE 2008;113:e2406. |
|17.||Heidari MR, Dadollahi Z, Mehrabani M, et al. Study of antiseizure effects of Matricaria recutita extract in mice. Ann N Y Acad Sci 2009;1171:300-4. |
|18.||Ogata I, Kawanai T, Hashimoto E, Nishimura Y, Oyama Y, Seo H. Bisabololoxide A, one of the main constituents in German chamomile extract, induces apoptosis in rat thymocytes. Arch Toxicol 2010;84:45-52. |
|19.||Hanigan M, Devaragan P. Cisplatin nephro-toxicity: moleular mechanisms. Cancer Ther 2003;1:47-61. |
|20.||Saad S, Najjar T, Daba M, Al-Rikabi A. Inhibition of nitric oxide synthase aggrevates cisplatin induced nephrotoxicity: Effect of 2-amino-4-methylpyridine. Chemotherapy 2002; 48:309-15. |
|21.||Al-Majed A, Abd-Allah A, Ammar C, Al-Rikabi A, Al-Shabanah O, Mostafa A. Effect of oral administration of Arabic gum on cisplatin-induced nephrotoxicity in rats. J Biochem Molecul Toxicol 2003;17:146-53. |
|22.||Kawai Y, Nakao T, Kunimura N, Kohada Y, Gemba M. Relationship of intracellular calcium and oxygen radicals to cisplatin related renal cell injury. J Pharmacol Sci 2006;100:65-72. |
|23.||Mohan I, Khan M, Shobha J, et al. Protection against cisplatin induced nephrotoxicity by spirulina in rats. Cancer Chemother Pharmacol 2006;58:802-8. |
|24.||Sadzuka Y, Shoji T, Takino Y. Mechanism of the increase in lipid peroxide induced by cisplatin in the kidneys of rats. Toxicol Lette 1992;62:293-300. |
|25.||Hara I, Miyake H, Yamanaka K, Hara S, Kamidono S. Serum cathepsin D and its density in men with prostate cancer as new predictors of disease progression. Oncol Rep 2002; 9:1379-83. |
|26.||Pompella A, Corti A, Paolicchi A, Giommarelli C, Zunino F. Gamma-glutamyltransferase, redox regulation and cancer drug resistance. Curr Opin Pharmacol 2007;7:360-6. |
|27.||Townsend D, Deng M, Zhang L, Lapus M, Hanigan M. Metabolism of cisplatin to a nephrotoxin in proximal tubule cell. J Am Soc Nephrol 2003;14:1-10. |
|28.||Viola H, Wasowski C, Levi De Stein M, et al. Apigenin, a component of Matricaria recutita flowers, is a central benzodiazepine receptors-ligand with anxiolytic effects. Planta Med 1995;61:213-6. |
|29.||Rekka E, Kourounakis A, Kourounakis P. Investigation of the effect of chamazulene on lipid peroxidation and free radical processes. Res Commun Mol Pathol Pharmacol 1996; 92:361-4. |
|30.||Krych M. Acute renal failure. Internist 2005; 46:30-8. |
Ragaa H.M. Salama
Associate Professor, Medical Biochemistry Department, Faculty of Medicine, Assuit University, Assiut
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]