Journal of
Toxicology and Environmental Health Sciences

  • Abbreviation: J. Toxicol. Environ. Health Sci.
  • Language: English
  • ISSN: 2006-9820
  • DOI: 10.5897/JTEHS
  • Start Year: 2009
  • Published Articles: 206

Full Length Research Paper

The possible protective effect of Zingiber officinale extract on cyclophosphamide-induced cardiotoxicity in adult male albino rats

Nashwa Mohamad M. Shalaby
  • Nashwa Mohamad M. Shalaby
  • Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Zagazig University, Egypt.
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Abeer Ramzy H. Mahmoud
  • Abeer Ramzy H. Mahmoud
  • Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Zagazig University, Egypt.
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Nagah El-Sayed M. Ali
  • Nagah El-Sayed M. Ali
  • 2Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Banha University, Egypt.
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Nahla E. Ibrahem
  • Nahla E. Ibrahem
  • Department of Histology, Faculty of Medicine, Zagazig University, Egypt.
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Noura Hussein Abdel H. Mekawy
  • Noura Hussein Abdel H. Mekawy
  • Department of Histology, Faculty of Medicine, Zagazig University, Egypt.
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  •  Received: 24 January 2019
  •  Accepted: 04 March 2019
  •  Published: 30 April 2019

 ABSTRACT

Cyclophosphamide, a cytotoxic alkylating agent, is an extensively used antineoplastic agent. Cardio-toxicity is considered as one of the limiting side effects of its use, which is attributed to oxidative stress. Zingiber officinale is powerful antioxidants against free radicals and oxidative attacks. The aim of this study is to investigate the possible protective effects of Z. officinale against cyclophosphamide induced cardio-toxicity in adult male albino rats. We used 30 adult male albino rats, divided into five groups; Group Ia (-ve control), Group Ib (+ve control), Group II (Z. officinale treated group; 200 mg/kg/day orally), Group III (cyclophosphamide treated group; single dose of 150 mg/kg I.P.), and Group IV (cyclophosphamide and Z. officinale treated group; rats received Z. officinale as before followed by single dose of cyclophosphamide (150 mg/kg)). At the end of experiment, we studied biochemical parameters: oxidative markers (MDA, GSH and Catalase), and Troponin i. The heart tissue was examined by light and electron microscope to evaluate histo-pathological changes and immune-histochemical technique for localization of inducible nitric oxide synthase (iNOS) in the cytoplasm of cardiomyocytes. The result showed increase in troponin I and disturbance of oxidative markers in cyclophosphamide treated group compared to control groups. Whereas these results had significantly improved in cyclophosphamide and Z. officinale treated group. Light and electron microscopic examination revealed disruption in the heart tissue histo-architecture in cyclophosphamide group with strong positive cytoplasmic iNOS immunoreaction in numerous cardiomyocytes by histochemical examination unlike that in cyclophosphamide and Z. officinale treated group which returned near normal. In conclusion, cyclophosphamide has a cardiotoxic effect which can be prevented by Z. officinale supplementation. Further studies about cyclophosphamide toxic effect on the heart and about Z. officinale role in protection are recommended.

Key words: Cyclophosphamide, Zingiber officinale, cardiotoxicty.

 


 INTRODUCTION

Cyclophosphamide is an alkylating agent having antineoplastic activity and immunosuppressive properties in many conditions like organ transplantation and systemic  lupus  erythematosus   (Nagler   et   al.,  2013).

Cardiotoxicity associated with high-dose cyclophosphamide has been described as a complication of several therapeutic regimens (Kamezaki et al., 2005) and the incidence of fatal cardiomyopathy varies from 2.0 to 17.0%, depending on the different regimens and patient populations (Taniguchi, 2005). It has been suggested that in order to benefit from the cyclophosphamide at higher doses, a protective agent was needed that would eliminate the toxic side effects of cyclophosphamide. Plasma antioxidant concentration has shown a decrease of patients who had a high dose chemotheraphy (Sabuncuoglu and Ozgunes, 2011).

Numerous studies had shown that cyclophosphamide exposure enhances intracellular reactive oxygen species (ROS) production, suggesting that biochemical and physiological side effects may result from its oxidative stress (Manda and Bhatia, 2003). Zingiber officinale (ginger), family Zingiberaceae is obtained from the underground stems or rhizomes of Zingiber. It is consumed as a fresh paste, dried powder, slices preserved in syrup, or candy or for flavoring tea. The underground stem or rhizome of this plant has been used as a medicine in Asian, Indian and Arabic herbal traditions since ancient times (Altman and Marcussen, 2001). It has been used in herbal medicine practice for the treatment of arthritis, rheumatologic conditions and muscular discomfort. Also, it has also been suggested for the treatment of various other conditions, including atherosclerosis, migraine headaches, rheumatoid arthritis, high cholesterol, ulcers, depression, and impotence. In addition to these medicinal uses, ginger continues to be valued around the world as an important cooking spice and is believed to help against common cold, flu-like symptoms, and even painful menstrual periods (Grant and Lutz, 2000). Its root contain polyphenol compounds (6-gingerol and shogaol), which have a high antioxidant activity (Stoilova et al., 2007). We aimed to investigate the possible beneficial protective effect of Z. officinale extract on cyclophosphamide induced cardiotoxicity in adult male albino rats.

 


 MATERIALS AND METHODS

Animals

In this study 30 male Sprague-Dawley albino adult rats, weighing 200–220 g, were used. Animals were fed ad libitum and housed in pairs in steel cages, having a temperature-controlled environment (22 ± 2°C) with 12 h light/dark cycles. Animal housing and handling were ethically considered. Painless procedures were conducted.

Chemicals

Cyclophosphamide was purchased from SIGMA-Aldrich (St. Louis, MO), HCY and CEPM were purchased from Santa Cruz Biotechnology (Dallas, TX).

Zingiber officinale: Fresh ginger (Z. officinale Roscoe, Zingiberacae) rhizomes were purchased from local commercial sources. The botanical authentication and extraction were done at the Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University.

Extraction of Z. officinale ethyl acetate extract

Fresh ginger rhizomes (400 gm) were divided, macerated and saturated with cold methanol (2 L) .The extract was filtered and subjected to drying. The residues were re-extracted for 3 times. The dried extract was dissolved in least amount of methanol (50 mL) and distilled water H2O was added, then fractionation was done by ethyl acetate using separating funnel at room temperature. Z. officinale ethyl acetate extract  was evaporated to dryness under reduced pressure at 40°C using rotatory evaporator (Hei- VAP Value Digital, Germany) (Lakshmi and Sudhakar, 2010).

Experimental protocol

The experimental animals were randomly divided into five equal groups each of 6 rats.

(i) Group Ia served as -ve control group and received regular diet and water for 10 days.

(ii) Group Ib (+ve control; gum acacia treated group): Each rat received a daily 0.5 ml gum acacia 1% aqueous solution (used for preparation of oral suspension of Z. officinale for 10 days by oral gavage.

(iii) Group II (Z. officinale treated group) (6 rats) Rats received a daily 200 mg/kg/day of Z. officinale for 10 days by oral gavage (El-Sharaky et al., 2009).

(iv) Group III (cyclophosphamide treated group) (6 rats) rats were injected intra-peritoneal with single dose of cyclophosphamide (150 mg/kg) (Gado et al., 2013).

(v) Group IV (cyclophsphamide and Z. officinale treated group) (6 rats) rats received Z. officinale as before followed by single dose of cyclophosphamide (150 mg/kg). Forty eight hour after cyclophosphamide treatment, under light ether anesthesia, venous samples from the retro-orbital plexus were obtained by capillary glass tubes as described by Schemere (1967). Rats were sacrificed and heart of each rat was dissected and grossly inspected to assess any gross abnormalities; then they were washed with cold normal saline and used for oxidative markers measurement, light and electron microscopic examination and immunohistochemical staining.

Biochemical parameters

Oxidative markers

(1) Determination of MDA (nmol/ g tissue: (MDA was assayed colorimetrically according to the method proposed by Ohkawa et al. (1979).

(2) Determination of GSH (mmol/g tissue): GSH was assayed colorimetrically according to the method proposed by Beutler et al. (1963).

(3) Determination of catalase activity (U/g): Catalase activity was assayed colorimetrically according to the method proposed by Aebi (1983).

Troponin i:  Was measured according to the method proposed by Collinson et al. (2001).

Light microscope examination

The heart was fixed in 10% formalin solution. After fixation, tissues were embedded in paraffin blocks and processed for 5 um thickness sections. These sections were stained by Hematoxylin and Eosin stains (Bancroft and Layton, 2013) and then examined by light microscope.

Immunohistochemical technique

It was used for localization of inducible nitric oxide synthase (iNOS) in the cytoplasm of cardiomyocytes. For the immunohistochemical staining of iNOS, paraffin sections were cleaned in xylene, hydrated, and then placed in phosphate buffered saline (PBS; pH 7.6). Sections were treated with 3% hydrogen peroxide and then washed with PBS. Sections were incubated, first with 1% pre-immune rabbit serum to decrease non-specific staining then with a monoclonal antibody against iNOS (1:200 dilutions; (clone 1A4, code No. 0034851). Detection of the antibody was performed using a biotin–streptavidin detection system. The positive results for iNOS immunoreactions were indicated by brown cytoplasmic staining (Kiernan, 2008; Bancroft et. al., 2013).

Electron microscope examination

Minute specimens were rinsed in 0.1M phosphate buffer saline (PBS)   pH 7.2 to remove blood from the surface.  heart tissues  greater  than  2  cm  long  were minced into smaller pieces of approximately 3  ×  3  mm  and  were  fixed  in  3% glutaraldehyde,   buffered   with   phosphate buffer  for  3  h.  It  was  rinsed  twice  with phosphate  buffer  for  10  min  per  rinse. The  tissues  were  then  fixed  in  2% aqueous  osmium  tetraoxide  for  2 h  and rinsed in 3 changes of distilled water for 10 min.  Each dehydration was accomplished   by immersion in a graded series of ethanol solutions of 25, 50, 75, 95 and 100%.  Infiltration with  propyleneoxide   and   embedding   with  increasing concentrations  of  propylene  oxide  was followed by  dehydration .  Thin  sections (600 nm)  were  obtained  by  use  of  ultra-microtome  and were  placed  on  a  copper 200  –  mesh  grid. They were stained with uranyl acetate and lead citrate and examined with GEOL-TEM1010 electron microscopic (Goodhew et al., 2003).

Histo-morphometrical analysis

The image analyzer computer system Leica Qwin 500 (Leica Ltd, Cambridge, UK) at the Image Analyzing Unit of the Pathology Department, Faculty of Dentistry, Cairo University (Egypt) was used to evaluate diameter of cadiac myocytes in H&E stained slides and optical  denisty  for  iNOS.  It  was  measured  using  the  interactive measure menu. Ten readings from five non-overlapping sections from each rat of all groups were examined.

Statistical analysis

The obtained data from biochemical (troponin i and oxidative stress markers (MDA & GSH & catalase activity) and morphometrical (diameter of cardiac myocytes and optical density of iNOS) analysis were expressed as mean ± SD (standard deviation) and subjected to one-way analysis of variance (ANOVA) and post hoc test using Statistical Package for the Social Sciences (SPSS) version 22.0. ANOVA was used for comparison between different groups (more than two groups), with p value less than 0.05 (the level of significance). Least significant difference (LSD) was used to find the statistical difference between the groups when ANOVA was statistically significant (P value <0.05) (SPSS Inc., 2013).

 


 RESULTS

Biochemical and statistical results

The biochemical findings among negative control, gum acacia and Z. officinale treated groups were statistically non-significant as regard all studied parameters (Table 1). While there were significant differences in the form of increase in troponin i and disturbance of oxidative markers in heart tissues in cyclophosphamide treated group when compared with the 1st three groups (-ve control, +ve control and Z. officinale treated group) (Table 1).

On supplementation of Z. officinale in (group IV), there was significant improvement of those parameters when compared with those in cyclophosphamide group (Group III).

 

 

Morphometrical and statistical results

Statistical analysis of the diameter of cardiac myocytes among negative control, gum acacia and Z. officinale treated groups, was not statistically significant. While, there was statistically significant decrease in diameter of cardiac myocytes of cyclophosphamide treated group (III) when compared with the previous groups (Table 2). A highly significant increase in the diameter of cardiac myocytes of cyclophosphamide and Z. officinale group (IV) was detected in comparison to that of cyclophosphamide treated group (III).

 

 

Statistical analysis of optical density of iNOS among negative, gum acacia and Z. officinale treated groups was not statistically significant as regard the studied parameters, while there was statistically significant increase of optical density of iNOS in cyclophophamide treated group (III) and a highly significant decrease in optical density of iNOS of cyclophophamide and Z. officinale group (IV) in comparison with cyclophophamide treated group (III) (Table 3).

 

 

Histopathological result

Light microscopic examination

H&E staining:  Histological  examination  of  H&E stained sections of control rats of left ventricular myocardium showed muscle fibers in different directions with acidophilic sarcoplasm, central pale oval nuclei. Delicate connective tissues separating  cardiac  myocytes were seen (Figures 1 and 2). While cyclophosphamide treated rats histopathogical examination revealed myocardial cell damage, necrosis and massive interstitial hemorrhage (Figure 3). Upon supplementation of Z.  officinale  there  was  partial  prevention  of the above pathological changes (Figure 4).

 

 

 

Immunohistochemical results: Immunohistochemical-stained sections of the control rats of left ventricular myocardium revealed weak positive cytoplasmic iNOS immunoreaction in cardiac myocytes (Figure 5). While, cyclophosphamide-treated rats showed strong positive cytoplasmic iNOS immunoreaction in numerous cardiac myocytes     (Figure     6).       On      the      other     hand, immunohistochemical-stained sections of cyclophosphamide and Z. officinale supplemented rats revealed moderate cytoplasmic iNOS immunoreaction in most of the muscle cells (Figure 7).

 

 

 

Transmission electron microscopic examination

An electron micrograph of a section from left ventricular myocardium  of a control group showed cardiac myocytes with euchromatic nucleus with dispersed chromatin.  Myofibrils revealed alternating dark and light bands and dark prominent Z-lines. Numerous mitochondria were seen  between the myofibrils.  Adjacent  cells  were connected by intercalated discs which appeared as dark irregular lines (Figure 8). While, an electron micrograph of a section from left venricular myocardium of cyclophosphamide treated group  showed  cardiac myocyte with some vacuolations. Nucleus with dispersed and peripheral chromatin condensation was observed (Figure 9). Upon supplementation of Z. officinale there was a partial prevention of these pathological changes (Figure 10).

 

 

 

 

 

 

 

 

 

 


 DISCUSSION

Cyclophosphamide is a nitrogen mustard alkylating agent with potent antineoplatstic, immunosuppressive, and immunomodulatory effects (Baba et al., 2012).

Cyclophosphamide is proved to be a cardiotoxic as it induced hemorrhagic myocarditis showing a typical clinical course invariably leading to mortality. Also, toxic endothelial damage by cyclophosphamide causes extravasation of toxic metabolites and results in myocyte damage, interstitial hemorrhage, and edema. The incidence of fulminant congestive heart failure is reported to be 5–19% (Kamezaki et al., 2005; Morandi et al., 2005). Cyclophosphamide cardiotoxic effect is related more to the magnitude of the dose administered during a single cycle rather than the cumulative dose over a period of time; cardiac failure associated with cyclophosphamide can be seen several days following administration (Dow et al., 1993).

Ginger (Z. officinale Rosco), a member of the family Zingiberaceae, well-known as a spice has been used for over 2000 years (Hasan et al., 2012). It contains biological active compounds as terpenes and oleoresin (Rahmani et al., 2014). Its broad spectrum of biological activities includes antioxidant, antimicrobial, antitumor or anti-diabetic effects (Masuda et al., 2004). It causes the suppression of both cyclooxygenase and lipoxygenase metabolites and arachidonic acid (Dugasani, 2010). The present study proved cardiotoxic effect of cyclophosphamide presented by significant increase in troponin I with disturbance of oxidative markers in heart tissue which was reversed significantly by supplementation of Z. officinale due to its antioxidant action. There was a significant decrease in the mean cardiac myocyte diameter in cyclophosphamide treated group as compared with all other groups. Some investigators have reported results similar to those of the current study (Iqbal et al., 2008).

According to Pai and Nahata (2000), high dose cyclophosphamide is associated with acute cardiotoxicity which is possibly related to an increase in free oxygen radicals. This increase would be mediated by elevated intracellular levels of the actual cytotoxic metabolite phosphoramide mustard. Cyclophosphamide administration has been proved to increase lipid peroxidation and deplete the antioxidant molecules as glutathione (GSH), catalase, and superoxide dismutase (Dorr and Lagel, 1994). Many studies have suggested that the use of antioxidants in combination with chemotherapy may prolong the survival of patients compared with expected outcome without antioxidant supplements (Manda and Bhatia, 2003; Sudharsan et al., 2006). These results are in line with Gado et al. (2013) who reported the possible cytoprotective effect of antioxidant against cyclophosphamide induced cardiotoxicity. Cetik et al. (2015) reported a dose-dependence on the cyclophosphamide -induced cardiotoxicity with disturbance of oxidative stress marker prevented by intake of carvacrol as antioxidant.

According to Chakraborty et al. (2017) combination of curcumin    with    piperine    exhibited   profound   cardio-protection effect against cyclophosphamide induced cardiotoxicity. Among the main active phytochemicals in Z. officinale gingerols, gingerdiol, shogaols, zingerone, and zingibrene are claimed to have antioxidant activity (Khaki et al., 2009). Some studies showed that Z. officinale treatment provided antioxidant effects by raising tissue concentrations of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). These antioxidants are important protection against oxidative stress due to their ability to detoxify free radicals, such as reactive oxygen species (ROS) (Harliansyah et al., 2005). Another study done by Morakinyo et al. (2010) supported the antioxidant property of ethanolic extract of Z. officinale. The present study revealed histopathological changes by both light and electron microscope in cyclophosphamide -treated rats which partially prevented upon supplemation of Z. officinale. These results are in line with Cetik et al. (2015) who reported histological findings as hemorrhagical lesions on myocardium and disruption of myocardial fibers prevented by intake of carvacrol as antioxidant.

Viswanatha-Swamy et al. (2013) proved massive change in the myocardium showing a varying degree of vacuolar changes in the cardiac muscle fibers mainly in the form of degeneration of myocardial tissue, vacuolization of the cardiomyocytes, infiltration of inflammatory cells and myofibrillar loss with cyclophosphamide treated group.

According to Omole et al. (2018) cyclophosphamide treated group shows distorted and wavy myocardial fibers with focal fatty change in some area of myocardial fibers. Loss of cellular constituents of the myocardial cells, myofibrillar loss and hypertrophic myocardial fiber with inflammation. improvement in histoarchitecture were noticed with use of Kolaviron as cardioprotective antioxidant. The result of immunohistochemical-stained sections of the control rats of the heart revealed weak positive cytoplasmic iNOS immunoreaction in cardiomyocytes. While, cyclophosphamide -treated rats showed strong positive cytoplasmic iNOS immunoreaction in numerous cardiomyocytes. On the other hand, immunohistochemical-stained sections of Z. officinale supplemented rats revealed moderate cytoplasmic iNOS immunoreaction in most of the muscle cells.

Inducible nitric oxide synthase is an enzyme induced by inflammatory cytokines or endotoxins, cellular disturbance and hypoxia as a defense mechanism. Cyclophosphamide -induced myocardial dysfunction led to hypoxia, with subsequent increased iNOS expression. This induction may be mediated by hypoxia inducible factor- 1α gene expression (HIF-1α) which is a transcriptional factor for iNOS. Several studies supported that hypoxia-induced up-regulation of iNOS expression in several cell types, including cardiomyocytes (Simone et al., 2010; Tekin et al., 2010).  

Fitzpatrick et al. (2005) stated that the up-regulation of iNOS expression is accompanied by increased nitric oxide (NO) production. The role of NO in cardiac function is complex, dependent on the level of NO produced.

Loren et al. (2009) mentioned that increased NO has been associated with relaxation of cardiac cells by an increase in cyclic GMP levels and likely plays an important role in modulating myocardial contractile function, ion channel activation, regulation of intracellular calcium, phosphorylation of contractile proteins.

 


 CONCLUSION

According to the results of this study, administration of cyclophosphamide in adult male albino rats induced cardiotoxicity at the histopathological and biochemical levels which can be alleviated by supplementation of Z. officinale extract.

 


 RECOMMENDATIONS

Human trials should be carried out, to establish the potential protective effects of Z. officinale in human intoxications. Future experiments are required to evaluate the possible protective molecular mechanisms of Z. officinale against cyclophosphamide -induced cardiotoxicity.

 


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.

 

 



 REFERENCES

Aebi H (1983). Catalase. In: Methods in enzymatic analysis, Bergmeyer HU (ed.), volume 3. Academic Press, New York pp. 276-286.

 

Altman RD, Marcussen KC (2001). Effects of a ginger extract on knee pain in patients with osteoarthritis. Arthritis and Rheumatism 44:2531-2538.
Crossref

 

Baba J, Watanabe S, Saida Y, Tanaka T, Miyabayashi T, Koshio J, Ichikawa K, Nozaki K, Koya T, Deguchi K, Tan C, Miura S, Tanaka H (2012). Depletion of radio-resistant regulatory T cells enhances antitumor immunity during recovery from lymphopenia. Blood 120:2417-2427.
Crossref

 

Bancroft J , Layton C (2013). The Hematoxylin and Eosin. Theory and Practice of histological techniques. 7th edition, pp. 106-115, 173-175 and 199-203. Elsevier, Churchill Livingstone, Philadelphia. 
Crossref

 

Beutler E, Duron O, Kelly MB (1963). Improved method for the determination of blood glutathione. Journal of laboratory and clinical medicine 61:882-888.

 

Cetik S, Ayhanci A, Sahinturk V (2015). Protective Effect of Carvacrol Against Oxidative Stress and Heart Injury in Cyclophosphamide-Induced Cardiotoxicity in Rat. Brazilian Archives of Biology and Technology 58:4. 
Crossref

 

Chakraborty M, Bhattacharjee A, Kamath JV (2017). Cardioprotective effect of curcumin and piperine combination against cyclophosphamide-induced cardiotoxicity. Indian Journal of pharmacology 49(1):65-70.

 

Collinson PO, Boa FG, Gaze DC (2001). Measurement of cardiac troponins. Annals of Clinical Biochemistry 38:423-449.
Crossref

 

Dorr RT, Lagel K (1994). Effect of sulfhydryl compounds and glutathione depletion on rat heart myocyte toxicity induced by 4- hydroperoxycyclophosphamide and acrolein in vitro. Chemico-Biological Interactions 93:117-28.
Crossref

 

Dow E, Schulman H, Agura E (1993) Cyclophosphamide cardiac injury mimicking acute myocardial infarction. Bone Marrow Transplantation 12:169-172.

 

Dugasani S, Pichika MR, Balijepalli MK, Tandra S, Korlakunta JN (2010). Journal of Ethnopharmacology 127:515-520.
Crossref

 

El-Sharaky AS, Newairy AA, Kamel MA, Eweda SM (2009). Protective effect of ginger extract against bromobenzene-induced hepatotoxicity in male rats. Food and Chemical Toxicology 47:1584-1590.
Crossref

 

Fitzpatrick CM, Shi Y, Hutchins WC, Su J, Gross GJ, Ostadal B, Tweddell JS, Baker JE (2005). Cardioprotection in chronically hypoxic rabbits persists upon exposure tonormoxia: role of nitric oxide synthase and KATP channels. American journal of physiology. Heart and Circulatory Physiology 288:62-68.
Crossref

 

Gado AM, Adam AN, Aldahmash BA (2013). Cardiotoxicity induced by Cyclophosphamide in rats:Protective effect of curcumin. Journal of Research in Environmental Science and Toxicology 2(4):87-95.

 

Goodhew PJ, Humphreys J, Beanland R (2003). Electron microscopy and analysis. In: Taylor and Francis. 4th ed., London, New York.

 

Grant KL, Lutz, RB (2000): Alternative therapies: ginger. American Journal of Health-System Pharmacy 57:945-947.
Crossref

 

Harliansyah AH, Noor Azian M, Zurinah WNW, Anum MYY (2005). Effects of zingiber officinale on superoxide dismutase, glutathione peroxidase, catalase, glutathione and malondialdehyde content in HepG2 cell line. Malaysian Journal of Biochemistry and Molecular Biology 11:36-41.

 

Hasan HA, Raauf AMR, Abd Razik BM, Hassan BAR (2012) Pharmaceutica Analytica Acta 3:2153-2435.

 

Iqbal M, Dubey K, Anwer T, Ashish A, Pillai KK (2008). Protective effects of telmisartan against acute doxorubicin-induced cardiotoxicity in rats. Pharmacological Reports 60:382-390.

 

Kamezaki K, Fukuda T, Makino S, Harada M (2005). Cyclophosphamide-induced cardiomyopathy in a patient with seminoma and a history of mediastinal irradiation. Internal Medicine 44:120-123.
Crossref

 

Khaki A, Fathiazad F, Nouri M, Afshin A (2009): The effects of ginger on spermatogenesis and sperm parameters of rat. Iranian Journal of Reproductive Medicine 7(1):7-12.

 

Kiernan JA (2008). Histological and Histochemical Methods. Theory and Practice. 4th edition. Butterworth Heinemann, Oxford, Boston pp. 129-139.

 

Lakshmi BVS, Sudhakar M (2010). Protective Effect of Zingiber officinale on Gentamicin-Induced Nephrotoxicity in Rats. International Journal of Pharmacology 6:58-62.
Crossref

 

Loren T, Yafeng D, Lashauna E (2009). Chronic hypoxia increases inducible NOS-derived nitric oxide in fetal guinea pig hearts. Pediatric Research 65(2):188-92.
Crossref

 

Manda K, Bhatia AL (2003). Prophylactic action of melatonin against cyclophosphamide-induced oxidative stress in mice. Cell Biology and Toxicology 19:367-372.
Crossref

 

Masuda Y, Kikizaki H, Hisamoto M, Nakatani N (2004). Antioxidant properties of gingerol related compounds from ginger. Biofactors 21:293-296.
Crossref

 

Morakinyo AO, Achema PU, Adegoke OA (2010). Effect of zingiber officinale (ginger) on sodium arsenite induced reproductive toxicity in male rats. African Journal Biomedical Research 13:39-45.

 

Morandi P, Ruffini PA, Benvenuto GM, Raimondi R, Fosser V (2005). Cardiac toxicity of high-dose chemotherapy. Bone Marrow Transplantation 35:323-334.
Crossref

 

Nagler A, Rocha V, Labopin M, Unal A, Ben Othman T, Campos A, Volin L, Poire X, Aljurf M, Masszi T, Socie G, Sengelov H, Michallet M, Passweg J, Veelken H, Yakoub-Agha I, Shimoni A, Mohty M (2013). Allogeneic hematopoietic stem-cell transplantation for acute myeloid leukemia in remission: comparison of intravenous busulfan plus cyclophosphamide (Cy) versus total-body irradiation plus Cy as conditioning regimen a report from the acute leukemia working party of the European group for blood and marrow transplantation. Journal of Clinical Oncology 31:3549-3556.
Crossref

 

Ohkawa H, Ohishi N, Yagi K (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95:351-358.
Crossref

 

Omole JG, Ayoka OA, Alabi QK, Adefisayo MA, Asafa MA, Olubunmi BO, Fadeyi BA (2018). Protective Effect of Kolaviron on Cyclophosphamide-Induced Cardiac Toxicity in Rats. Journal of Evidence-Based Integrative Medicine 23:1-11.
Crossref

 

Pai VB, Nahata MC (2000). Cardiotoxicity of chemotherapeutic agents. Incidence, treatment and prevention. Drug Safety 22(4):263-302.
Crossref

 

Rahmani AH, Al shabrmi FM, Aly SM (2014). Active ingredients of ginger as potential candidates in the prevention and treatment of diseases via modulation of biological activities. International Journal of Physiology, Pathophysiology and Pharmacology 6:125-136.

 

Sabuncuoglu S, Ozgunes H (2011). Chemotherapy, free radicals and oxidative stress. Hacettepe University Journal of the Faculty of Pharmacy 31(2):137-150.

 

Schemere S (1967). The blood morphology of laboratory animals, 3rd ed., C.V. Mosby Co, pp. 10- 65.

 

Simone R, Subash C, Gupta N, Madan M, Chaturvedi C, Bharat B, Aggarwal S (2010). Oxidative stress, inflammation, and cancer: How are they linked? Free Radical Biology and Medicine 49(11):1603-1616.
Crossref

 

SPSS Inc (2013). SPSS for windows, version 22.0. Chicago, SPSS Inc. 

 

Stoilova I, Krastanov A, Stoyanova A, Denev P, Gargova S (2007): Antioxidant activity of a ginger extract (Zingiber officinale). Food Chemistry 102: 764-770. 
Crossref

 

Sudharsan PT, Mythili Y, Selvakumar E, Varalakshmi P (2006). Lupeol and its ester meliorate the cyclophosphamide provoked cardiac lysosomal damage studied in rat. Molecular and Cellular Biochemistry 282:39-44.
Crossref

 

TaniguchiI (2005). Clinical significance of cyclophosphamide-induced cardiotoxicity. Internal Medicine 44:123-35.
Crossref

 

Tekin D, Dursun AD, Xi L (2010). Hypoxia inducible factor 1 (HIF-1) and cardioprotection. Acta pharmacologica Sinica 31(9):1085-1094.‏
Crossref

 

Viswanatha SAHM, Patel UM, Koti BC, Gadad PC, Patel NL, Thippeswamy AHM (2013). Cardioprotective effect of Saraca indica against cyclophosphamide induced cardiotoxicity in rats: A biochemical, electrocardiographic and histopathological study. Indian Journal of Pharmacology 45(1):44-48.
Crossref

 




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