RJPS Vol No: 14 Issue No: 1 eISSN: pISSN:2249-2208
Lolita Prima Fernandes*, Ben Mathew, Jagadish V Kamath
Department of Pharmacology, Shree Devi College of Pharmacy, Mangalore-574142, Karnataka
Author for Correspondence
Lolita Prima Fernandes
Asst. Professor
Department of Pharmacology
Shree Devi College of Pharmacy
Mangalore-574142, Karnataka, India
Email: lolitaprima58@gmail.com
Abstract
The present study was undertaken to evaluate the effect of Simarouba glauca leaf extract (SGLE) on Cyclophosphamide (CYP) induced cardiotoxicity in experimental animals.In CYP induced cardiotoxicity model, rats of either sex were treated with CYP (200 mg/kg, i.p) on the first day of experimental period with leaf extract of Simarouba glauca (400 mg/kg and 200 mg/kg, p.o) for 10 days. The influence of the treatment was analyzed by quantification of biomarkers, antioxidants, electrocardiogram (ECG) parameters, and histopathological observation.In the case of CYP induced cardiotoxicity model, the activities of superoxide dismutase (SOD) and catalase (CAT) were increased and creatinine kinase-MB (CK-MB), creatinine kinase-NAC (CK-NAC), lactate dehydrogenase (LDH), lipid peroxidation (LPO) were decreased in serum of Simarouba glauca treated groups when compared to CYP control group. Similarly, the ECG changes were restored close to normal in Simarouba glauca treated groups.Thus, investigational finding concluded that Simarouba glauca leaf extract possesses potential benefits against myocardial damage induced by anticancer drug Cyclophosphamide
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INTRODUCTION
Selectivity of majority of anti-cancer drugs is limited due to their deliberate adverse effect in cancer therapy1 . Cancer is a broad group of various diseases, medically known as a malignant neoplasm which is characterized by uncontrolled growth of the cells, forming malignant tumors and invade near by parts of the body through the lymphatic system or blood stream.2
Chemotherapy is one of the major treatment approaches for cancer.3 Commonly used cytotoxic drugs includes doxorubicin, cisplatin, methotrexate, cyclophosphamide, mechlorethamine, and dacarbazine and antiangiogenesis drugs.4
Cyclophosphamide is nitrogen mustard type alkylating agent, which is one of the most effective anticancer drugs widely used for the treatment of breast, lung, ovarian, bladder, and brain cancer. Unfortunately, the clinical usefulness of cyclophosphamide is usually limited by the development of undesirable side effects such as nephrotoxicity, cardiac necrosis, hepatotoxicity, anaphylactic reaction, etc.5
Many plants are reported to have protective capacity towards the toxic effects of many drugs and pharmaceuticals. Some of the herbs like Withania somnifera and Zingiber officinalis are examples of such plants. These plants exhibit potent antioxidant effects due to the phytoconstituents like triterpenoids, polyflavonoids.6,7
Simarouba glauca, belongs to family Simaroubaceae, is a rain fed wasteland evergreen edible oil tree, commonly known as ‘Laxmitaru’ or ‘Paradise tree’.8 It is used as anti-inflammatory, anti-allergic, and anti-carcinogen. The plant also shows antiviral, antioxidant, and myocardial activities.9 The main active group of phyto chemicals in Simarouba are the quassinoids, which belong to the triterpene chemical family.10 Ailanthinone, glaucarubinone and holacanthone are considered some of the main active quassinoids in Simarouba.11
The effect of Simarouba glauca on cyclophosphamide induced toxicity on heart are not yet proved. Thus, current study was focused on the effect of leaf extract of Simarouba glauca on cyclophosphamide induced cardiotoxicity in experimental animals.
MATERIALS AND METHODS
Isolation of Simarouba glauca and it’s dose selection
The leaves of Simarouba glauca were collected from different parts of Kerala. The leaves collected were washed thoroughly in water, chopped and air dried for a week at 35-40 ºC. Then chopped leaves were pulverized in electric grinder and extracted in Soxhlet apparatus, using petroleum ether and ethanol. The extract was concentrated under reduced pressure and made to powder. These powders were dissolved in DMSO, then administered orally to the animals by using oral feeding needle.10 Based on earlier literature review, lethal dose (LD50) of the extract was found to be 2000 mg/kg in albino rats, hence in the present study therapeutic doses were selected as 200 mg/ kg and 400 mg/kg, p.o.11
Experimental animals
Albino wistar rats of either sex weighing 170- 250 g were housed in standard polypropylene cages and maintained under controlled room temperature (25 ± 5°C) and relative humidity (55 ± 5%) in a well-ventilated animal house under 12:12 h light and dark cycle. All the rats were provided with commercially available standard pellet diet, water ad libitum. The animals were maintained under standard conditions in theanimal house as per the guidelines of Committee for the Purpose of Control andSupervision on Experiments on Animals (CPCSEA). The Institutional Animal Ethics Committee approved the experimental protocol (SDCP/IAEC-24/2014-15).
Cyclophosphamide induced cardiotoxicity
In this experimental model,albino wistar rats of either sex were divided in to 5 groups of 6 animals each.
Group- I : Control (1mL/kg of 0.5% carboxy methyl cellulose).
Group –II : CYP (200mg/kg, i.p.) serves as toxic control.
Group -III : Ethanolic extract of S.glauca (400mg/kg).
Group- IV : Ethanolic extract of S.glauca (400mg/kg BW) +CYP (200mg/kg).
Group- V : Ethanolic extract of S.glauca (200mg/kg BW) + CYP (200mg/kg).
Group I served as normal control (1 mL/ kg of 0.5% carboxy methyl cellulose) for 10 days. Group II served as cyclophosphamide control where animals received a single dose of cyclophosphamide (200 mg/kg, I.P.) on the first day of experimental period. Group III animals received ethanolic extract of Simarouba glauca (400 mg/kg, P.O.) alone for 10 days. Group IV animals received single dose of cyclophosphamide
(200 mg/kg i.p.) on first day followed by the administration of ethanolic extract (400 mg/kg) of Simarouba glauca continuously for 10 days. Group V animals daily received Simarouba glauca ethanolic extract (200 mg/kg) for 10 days immediately after a single dose of cyclophosphamide (200 mg/kg i.p.) on first day.12
Parameters used for assessment of toxicity
Biomarkers level such as creatinine kinase isoenzyme MB (CK-MB), Creatinine kinase N-acetyl cysteine (CK-NAC) and Lactate dehydrogenase (LDH).13
Antioxidant level such as Superoxide dismutase (SOD),14 Catalase15 and Lipid peroxidation.16
ECG recordings: Heart rate, QRS interval, PR interval, QT segment and RR interval.17,18
Histopathological studies.19
Statistical analysis
Results are expressed as Mean ± SEM. Statistical significance was assessed using One-way Analysis of variance (ANOVA) followed by Tukey-Karmer multiple comparison tests. p<0.05 was considered significant.
RESULTS
Effect of Simarouba glauca on cyclophosphamide induced cardiotoxicity
Effect on electrocardiographic parameters
Effect on heart rate: Electrocardiographic determination revealed an extremely significant (p<0.001) decrease in heart rate of CYP control group compared to normal control group.Prior treatment with S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001) rise in heart rate values compared to CYP control.
Effect on QRS duration: An extremely significant (p<0.001) increase in QRS duration was observed in CYP control group compared to normal control. Treatment of animal with S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001) decrease in QRS duration compared to CYP control group.
Effect on QT segment: CYP control group showed an extremely significant (p<0.001) increase in QT segment compared to normal control.Prior treatment of S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001) decrease in QT segment compared to CYP control group.
Effect on RR interval: CYP control group showed an extremely significant (p<0.001) increase in RR interval compared to normal control.Treatment of animal with S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001) decrease in RR interval compared to CYP control group.
Effect on PR interval: CYP control group showed an extremely significant (p<0.001) increase in PR interval compared to normal control. Treatment of animal with S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001) decrease in PR interval compared to CYP control group.
All the values are in Mean±SEM, n=6, ns= not significant, *p<0.05, **p<0.01,***p<0.001 when compared to normal, #p <0.05,##p<0.01,###p<0.001 when compared to CYP control
Effect on serum biomarkers:
Effect on serum CK-MB level: An extremely significant (p<0.001) increase was observed in CKMB level in CYP control group compared to normal control. Prior treatment of S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001 and p<0.05) decrease in CK-MB compared to CYP control group, respectively.
Effect on serum CK-NAC level: An extremely significant (p<0.001) increase was observed in CK-NAC level in CYP control group compared to normal control. Prior treatment of S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001) decrease in CKNAC compared to CYP control group.
Effect on serum LDH level: An extremely significant (p<0.001) increase was observed in LDH level in CYP control group compared to normal control. Prior treatment of S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001) decrease in LDH compared to CYP control group.
All the values are in Mean±SEM, n=6, ns= not significant,*p<0.05,**p<0.01,***p<0.001 when compared to normal, #p <0.05,##p<0.01,###p<0.001 when compared to CYP control.
Effect on antioxidants
Effect on SOD : Antioxidant study on the heart tissue homogenate showed an extremely significant (p<0.001) decrease in SOD level in CYP control group compared to normal control.Prior treatment of S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001) increase in SOD level compared to CYP control.
Effect on catalase : Antioxidant study on the heart tissue homogenate showed an extremely significant (p<0.001) decrease in catalase level in CYP control compared to normal control. Prior treatment of S.glauca (400mg/kg) and S.glauca (200mg/kg) showed an extremely significant (p<0.001) increase in catalase level compared to CYP control.
Effect on lipid peroxidation: The CYP control (200mg/kg, i.p) illustrated an extremely significant (P<0.001) increase in lipid peroxidation when compared to normal control group. Treatment of S.glauca (400 mg/kg) and S.glauca (200 mg/kg) demonstrated a moderately significant (p<0.01) and an extremely significant (p<0.001) decrease in lipid peroxidation, respectively when compared with the CYP control group.
All the values are in Mean±SEM, n=6, ns= not significant,*p<0.05,**p<0.01,***p<0.001 when compared to normal, #p <0.05,##p<0.01,###p<0.001 when compared to CYP control.
Histopathological observation
The stained microscopic section of normal control group showed the normal structure of heart. The cardiac muscle fibres were found to be uniform size, shape, and configurations with no inflammatory cell infiltrates.
Cyclophosphamide (200mg/kg, i.p.) control group showed distorted structure of heart by necrosis of the cells with degeneration of myofibril, increased interstitial space, and diffused inflammation.
S.glauca treated group showed normal structure of heart, which indicate that the leaf extract of S.glauca did not possess any toxic effect on heart.
CYP + S.glauca 400mg/kg showed effective inhibition of CYP induced cardiac damage by reversal of infiltration of inflammatory cells and fragmentation of myofibrils.
CYP + S.glauca 200mg/kg showed moderate myocardial damage with less interstitial space and myofibrillar degeneration.
Histo pathological slides of rat heart tissue in CYP induced cardiotoxicity
DISCUSSION
The current research was designed to evaluate the effect of Simarouba glauca leaf extract on cyclophosphamide induced cardiotoxicity in experimental animals. The study revealed a highly significant protective action of Simarouba glauca leaf extract against myocardial injury caused by the anticancer drugs.
Simarouba glauca commonly known as “lakshmi taru” or “paradise tree” belongs to the family Simaroubaceae.20 The preliminary phytochemical estimation has revealed the presence of alkaloids, carbohydrates, reducing sugar, saponins, proteins, tannins, flavonoids, and glycosides. Extract of this herb had been proven to have therapeutic effects in many clinical studies. It includes antimicrobial, insecticidal, febrifuge, antidysenteric, antiherpetic, antihelminthic, antiulcer, and antiprotozoal activities.11 The leaf, fruit, pulp, and seed show properties such as analgesic, antimicrobial, antiviral, astringent, emmenagogue, stomachic, tonic, vermifuge.21 Cardiotoxicity was induced in rats by administering CYP (200mg/kg, i.p)12 onthe first day of experimental period. After an experimentation period that last for 10 days, they all were dispatched and different parameters were evaluated as per the protocol.Cardiotoxicity was caused due to the destructive effect of CYP. CYP mediates production of xanthine oxidase which catalyses the oxidation of hypoxanthine to xanthine and generates superoxide and uric acid. Superoxide is known to generate reactive oxygen species. Thus, it can be understood that CYP causes increased production of free radicals and decreased levels of anti-oxidant enzymes. It also induces lipid peroxidation.22
In our present finding, animals treated with only CYP demonstrated significant decrease in SOD, catalase and significant increase in lipid peroxidation, which indicates the induction of myocardial toxicity. Prophylactic treatment with Simarouba glauca dose dependently increase SOD and catalase activity and decrease lipid peroxidation level which justify it’s protection.
CYP induced myocardial damage is responsible for leakage of marker enzymes such as CK-MB, CK-NAC, and LDH from the myocytes to the blood. Estimation of these marker enzymes serves as a diagnostic tool to detect myocardial necrosis.23
In the present study, experimental animals treated with CYP without any other treatment reflected remarkable amount of increase in serum marker enzymes levels which confirms the induction of myocardial toxicity. Simarouba glauca dose dependently restored the marker enzymes levels.
In the present study, CYP showed abnormal changes in electrocardiographic pattern such as decrease in heart rate and increase in RR, PR, and QT intervals and prolongation of QRS interval. Abnormal changes in ECG parameters were also found to be extremely significant (p ≤ 0.001) compared to normal control.
Decrease in heart rate may be due to release of significant amount of acetylcholine, which is also linked with the genesis of myocardial damage.24 Prolongation of QT interval found in present study with CYP may be due to increase in the cellular Na+ content and decrease in K+ content. Hypokalemia is suspected for QT interval elongation. Prolongation of PR interval associated with CYP might be due to AV block. Change in parasympathetic tone and conduction system deformation can be the reason for AV block.25
Simarouba glauca in both 400mg/kg and 200mg/ kg doses, in a dose dependent manner bring back the ECG parameters towards the normal.
Histological study in CYP induced cardiotoxicity supported the findings of other parameters analyzed in different treatment groups. For the normal heart, myocardial fibers were found to be of uniform size, shape, and configurations with no inflammatory cell infiltrates. Treatment with Simarouba glauca dose dependently inhibited CYP induced cardiac damage by decreasing fragmentation of myofibrils and inflammation. Simarouba glauca predominantly in higher dose (400 mg/kg) was able to retrieve the pathological changes associated with CYP in myocardial cell.
CONCLUSION
The present investigation demonstrated the effect of Simarouba glauca leaf extract on cyclophosphamide (CYP) induced nephrotoxicity in experimental animals. In CYP induced toxicity model; prophylactic treatment of Simarouba glauca (400mg/kg and 200mg/kg, i.p) showed cardio protection by showing extremely significant (p<0.001) effect. The efficacy of the herb could be mainly attributed to its potential antioxidant property and free radical scavenging activity. Further research is required to establish the fact clinically.
CONFLICTS OF INTEREST
The authors declare no conflicts of interests.
Supporting Files
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