Cardioprotective effect of methanolic extract of Ixora coccinea Linn. leaves on doxorubicin-induced cardiac toxicity in rats.

OBJECTIVES: To investigate the effect of methanolic extract of Ixora coccinea Linn. (MEIC) leaves against doxorubicin-induced cardiac toxicity in rats.
MATERIAL AND METHODS: Albino Wistar rats were pretreated with the methanolic extract of Ixora coccinea Linn. leaves (200 and 400 mg/kg, orally) for 1 week followed with the simultaneous treatment with doxorubicin (cumulative dose of 15 mg/kg in six divided doses for 2 weeks) along with the extracts for the next 14 days. On the 22(nd) day hemodynamic parameters such as blood pressure and ECG were recorded. Biochemical study including biomarkers like creatine kinase - MB (CK - MB), lactate dehydrogenase (LDH), SGOT and SGPT, tissue antioxidant markers viz. catalase (CAT), superoxide dismutase (SOD) and extent of lipid peroxidation viz. malondialdehyde (MDA) was estimated. Histopathology of heart was also done to assess the cardioprotective effect.
RESULTS: Pretreatment with MEIC significantly reduced (P<0.01) the ST segment elevation and also maintained the BP (P<0.01) close to normal. The MEIC significantly reduced the elevated level of biomarkers like CK - MB, LDH, SGOT, SGPT (P<0.01) near to normal, the MEIC also increased the tissue antioxidant markers viz. CAT, SOD and decreased the level of MDA (P<0.01) in cardiac tissue by dose-dependant manner. The histopathology of heart also further confirmed the cardioprotection provided by the methanolic extract of Ixora coccinea Linn. leaves.
CONCLUSION: The results suggest a cardioprotective effect of Ixora coccinea Linn. leaves due to its antioxidant properties.

Introduction

Ixora coccinea Linn. (Fam: Rubiaceae) is a shrub with a small, obovate to oval–oblong, rounded to subcordate base leaves on branched hard heavy twigs. Height commonly goes to around 4 – 6 ft. (1.2 – 2 m) but capable of going upto 12 ft (3.6 m) Ixora coccinea Linn. is found throughout India more common in the western peninsula in scrub jungles widely cultivated throughout the tropics. Roots are used as sedative for hic - coughs, used for nausea, loss of apetite, fever, gonorrhea, diarrhea, decoction is given for dysentry. Leaves are used for dermatological disorders, methanolic extract of Ixora coccinea Linn. leaves has shown antioxidant and reactive oxygen species scavenging property, moreover Ixora coccinea Linn. extract showed strong reducing power and total antioxidant capacity. Leaf and stem are used as an ablution for infantile. Flowers and bark is used on reddened eyes and eruptions in children. Decoction of flowers is given for hemoptysis, catarrhal bronchitis and dysmenorrhea. Flowers are applied externally to sores, employed for chronic ulcer, scabies and some type of dermatitis and also used internally for cholera, diarrhea, dysentry, leucorrhea, antitumor and gonorrhea, fresh juice of flower have protective action against electroconvulsions.[1-3] Ixora coccinea Linn. flowers showed chemoprotective effects on cyclophosphamide-induced toxicity by increasing the life span of treated mice[4] and also on cisplatin-induced toxicity.[5] Ether and methanolic extract of Ixora coccinea Linn. dry leaves have antimicrobial activity. Aqueous extract of Ixora coccinea Linn. leaves have showed antinociceptive activity in mice.[6]

Doxorubicin (Dox) is a well-known antineoplastic drug, the use is limited due to potential of causing myocardial damage. The mechanism by which DOX acts is by the formation of an iron-anthracycline complex that generates free radicals, which in turn, causes severe damage to the plasma membrane, and interferes with the cytoskeletal structure.[7] Oxygen free radical formation by doxorubicin[89] enhances the susceptibility of cardiac tissue to lipid peroxidation leading to a progressive dose–related irreversible loss of myofibrils, dilation of the sarcoplasmic reticulum, cytoplasmic vacuolization, swelling of mitochondria, increased number of lysosomes and myocyte necrosis,[10] inhibition of nucleic acid as well as protein synthesis,[11] release of vasoactive amines,[12] change in adrenergic function,[13] decreased activity of Na[+] K[+] ATPase,[14] alteration in sarcoplasmic calcium transport and imbalance of myocardial electrolytes in response to the doxorubicin.[15]

As there were no previous reports available for the cardioprotective activity of the drug, the present study was undertaken to study the cardioprotective effect of methanolic extract of Ixora coccinea Linn. leaves on Doxorubicin (Dox)-induced cardiac toxicity with a view to provide a scientific evidence.

Materials and Methods

Plant Material

The leaves of Ixora coccinea Linn. were collected in the month of August 2010 from local area of Sangli, Maharashtra and were authenticated by Dr. (Mrs.) U. S. Yadav, Dept. of Botany, Willingdon college, Sangli. The herbarium has been submitted for further reference (V. No. WC/2010/121). The plant material was shade dried at room temperature.

Preparation of Extract

The dried leaves of Ixora coccinea Linn. were ground and made into a coarse powder. The coarse powder was then subjected to continuous hot extraction in soxhlet apparatus with methanol as a solvent and extracted till the solvent becomes colorless. The extract was evaporated under reduced pressure using rotary evaporator (Equitron Roteva) at a low temperature of 45°C until the extract turned syrupy and then the syrupy extract was transferred to an evaporating dish for drying at room temperature. The yield was found to be 16% w/w.

Preliminary Phytochemical Investigation

The extract was subjected to chemical tests qualitatively for identification of different phytoconstituents like glycosides, saponins, carbohydrates, sterols, alkaloids, flavonoids, tannins, proteins, triterpenoids.

Animals

Albino Wistar rats of 150-250 g of either sex were used for the study. The inbred species of rats were obtained from animal house of Appasaheb Birnale College of Pharmacy, Sangli, for experimental purpose. The animals were maintained under controlled conditions of temperature (23 ± 2°C), humidity (50 ± 5%) and 12-h light - dark cycles. All the animals were acclimatized for seven days before the study. The animals were randomized into experimental, normal and control groups, housed individually in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free access to standard pellets as basal diet and water ad libitum. Animals were habituated to laboratory conditions for 48 h prior to experimental protocol to minimize if any of non-specific stress. All the studies conducted were approved by the Institutional Animal Ethical Committee (IAEC) of Appasaheb Birnale College of Pharmacy, Sangli, Maharashtra (Registration No. 843/AC/04/CPCSEA), India.

Toxicity Studies

Acute toxicity study was carried using albino rats by up and down/staircase method as per OECD guidelines. The methanolic extract of Ixora coccinea Linn. (MEIC) was orally administered to different groups of rats at the doses of 50, 300, 1000, 2000 and 3000 mg/kg body weight, respectively. The animals were observed for 48 hr to study the general behavior and for any sign of discomfort to the animals. There was no mortality found upto dose 3000 mg/kg.

Chemicals

Sodium carboxy methyl cellulose (Na - CMC), thiobarbituric acid, heparin, ketamine (Neon Labs, India), xlyazine (Indian Immunologicals, India), doxorubicin (Cipla, India), sodium hydroxide, pyridine, n – butanol, pyrogallol, anesthetic ether, etc., the enzyme kits used were creatine kinase – MB (Pathozyme, India), lactate dehydrogenase (Teco diagnostics, India), SGOT (Span diagnostics Ltd., India), SGPT (Span diagnostics Ltd., India), total protein (Pathozyme, India) etc., all the chemicals used was of analytical grade.

Doxorubicin-induced cardiac stress

The rats were divided into four groups of six animals each. The groups were as follows:

Group 1: Normal (rats treated with 1% Na CMC, ~2 ml/kg/day, p.o). Group 2: Control (rats treated with Dox with total cumulative dose of 15 mg/kg i.p. for 2 weeks in six divided dosage).[15] Group 3: Rats pretreated with methanolic extract of Ixora coccinea Linn. (MEIC) 200 mg/kg p.o. along with Dox treatment. Group 4: Rats pretreated with (MEIC) 400 mg/kg p.o. along with Dox treatment.

Group 2, 3 and 4 received Dox at alternate days for a period of 2 weeks (the days selected for Dox injection was on the 8th, 10th, 14th, 16th, 18th, 21st day after the 7 days pretreatment with the extract.

On the 22nd day parameters studied were general appearance, heart weight and heart/body weight ratio. Electrocardiography (ECG) was recorded at the end of the treatment after the last dosing of Dox. All rats were fasted overnight but had free access to water after the last dose administration. Biopac MP - 35 (Santa Barbara, CA, USA) was used to record and monitor ECG tracings. Rats from each group were anesthetized with Ketamine – Xylazine anesthesia, a needle electrodes were inserted under the skin for the limb lead at position II. For each ECG tracing P wave, QRS complex, QT interval, RR interval and cardiac cycle were measured.

Blood Pressure

Blood pressure was determined by invasive method (carotid artery cannulation) by using BIOPAC MP – 35. Carotid artery was cannulated by using PE – 50 tube which was attached to the pressure transducer loaded previously with the heparinized saline. Here, various parameters such as systolic, diastolic, mean BP and heart rate was determined.

Biochemical parameters

Soon after the blood pressure had been recorded, the blood was collected from the retro orbital route and subjected to centrifugation to isolate serum out of it. The serum was further used for estimation of creatine kinase – MB (CK - MB) by immunoinhibition method estimated at 340 nm, lactate dehydrogenase (LDH) by kinetic method estimated at 340 nm, serum glutamate oxaloacetate transaminase (SGOT)/aspartate aminotransferase (AST) and serum glutamate pyruvate transaminase (SGPT)/alanine aminotransferase (ALT) by 2,4-DNPH method estimated at 505 nm. Heart tissue homogenate was prepared in 0.05 M phosphate buffer pH 7.4 and homogenated in tissue homogenizer at 2000 rpm for 10 min. Antioxidant enzymes estimated were catalase[16] (CAT), superoxide dismutase[17] (SOD) and extent of lipid peroxidation malondialdehyde[18] (MDA).

Histopathological study

At the end of study, the heart was isolated, washed with ice cold saline. The tissue was fixed in 10% buffered neutral formalin solution. After fixation, tissues were embedded in paraffin - wax and sections were cut and stained with hematoxylin and eosin. The slides were observed under light microscope.

Statistical Analysis

Values are expressed as Mean±SEM for six rats in each group, statistical analysis was performed using one way ANOVA followed by Dunnett t test (GraphPad Instat, USA). **P<0.01 was taken as the criterion of statistical significance.

Results

Chronic administration of Dox-induced cardiac toxicity and the effect of methanolic extract of Ixora coccinea Linn. leaves was established by assessing the hemodynamic, electrocardiographic, biochemical and histopathological studies.

Preliminary phytochemical investigation revealed the presence of steroids, tannins, flavonoids, glycosides and alkaloids.

Behavior Changes

No behavior changes were observed during the toxicity studies.

General observation

In Dox-treated group, the animal fur became scruffy and developed a yellowish to reddish tinge. These rats also had red exudates around the eyes and nose, soft watery feces, animals were sicker and lethargic when compared with the normal. These observations were markedly reduced in the group treated with MEIC when compared with the doxorubicin control group.

Body weight and heart/body weight ratio

In Dox-treated rats, decrease in body weight, heart weight and heart/body weight ratio was seen at the end of the study when compared with the normal. MEIC 200 and 400 mg/kg, dose dependently demonstrate significant increase in body weight gain, increase in weight of heart (MEIC – 200 mg/kg, **P<0.01) and increase in heart/body weight ratio (**P<0.01), when compared with Dox control group, [Table 1].

Table 1

Effect of methanolic extract of Ixora coccinea Linn. (MEIC) leaves on body weight, heart weight and heart/body weight ratio by doxorubicin-induced cardiotoxicity

ECG recordings

The electrocardiographic pattern of normal, control, MEIC 200 and 400 mg/kg is shown in Figure 1. Normal group showed a normal ECG pattern, where as animals treated with Dox alone showed significant elevation in ST segment, prolongation in P wave, QRS complex and R - R interval. In addition there was a decrease in cardiac cycles and prolongation of QT interval as compared to normal rats. Treatment with MEIC 200 and 400 mg/kg for 21 days alternatively with Dox-treated rats exhibited near to normal ECG pattern with a decreased elevation in ST segment. Furthermore, treatment also resulted in decrease in P wave (MEIC - 400 mg/kg, **P<0.01), QRS complex (MEIC, 400 mg/kg, **P<0.01), QT interval (MEIC – 200 and 400 mg/kg, **P<0.01) and R – R interval (MEIC 200 and 400 mg/kg, **P<0.01), whereas cardiac cycle was increased (MEIC – 200 and 400 mg/kg, **P<0.01) when compared with the control. The data such as P wave, QRS complex, QT interval, R – R interval and cardiac cycle are shown in Table 2.

Figure 1

ECG recordings of normal, control (Dox treated), MEIC 200 mg/kg, MEIC 400 mg/kg. The green highlighted part of normal ECG shows normal ST segment. The blue circle indicates ST segment elevation of doxorubicin control group. Groups treated with MEIC 200 mg/kg and 400 mg/kg shows close to normal ST segment indicated by green and brown color circle, respectively, when compared with doxorubicin control group

Table 2

Effect of methanolic extract of Ixora coccinea Linn. (MEIC) leaves on ECG changes (in seconds) of rats by doxorubicin-induced cardiac toxicity

Blood pressure determination

The animals treated with Dox showed a decrease in the systolic, diastolic, mean BP and the heart rate when compared with the normal, treatment with methanolic extract of Ixora coccinea Linn. leaves 200 mg and 400 mg/kg showed a dose-dependent, significant increase in the systolic BP (**P<0.01), diastolic BP (**P<0.01), mean BP (**P<0.01) and the heart rate (**P<0.01), respectively, when compared with the control group. Table 3 shows the data for blood pressure.

Table 3

Effect of methanolic extract of Ixora coccinea Linn. (MEIC) leaves on blood pressure (mmHg) of rats by doxorubicin-induced cardiac toxicity

Serum Markers: CK – MB, LDH, SGOT and SGPT

Treatment with doxorubicin causes an elevation in level of these enzymes which are considered as the selective biomarkers of myocardial damage when compared with the normal. Our study showed decrease in the elevated levels of these enzymes. Pretreatment with MEIC 200 mg and 400 mg/kg showed a dose dependent significant decrease in CK - MB (**P<0.01), LDH (**P<0.01), SGPT (**P<0.01) and SGOT (**P<0.01) when compared with the Dox control group. Table 4, shows the effect of methanolic extract of Ixora coccinea Linn. leaves 200 mg and 400 mg/kg on the level of creatine kinase – MB, lactate dehydrogenase, SGOT and SGPT enzymes.

Table 4

Effect of methanolic extract of Ixora coccinea Linn. (MEIC) leaves on CK – MB, LDH, SGOT and SGPT levels in rats by doxorubicin-induced cardiac toxicity

Tissue antioxidant markers and lipid peroxidation of heart tissue homogenate

Doxorubicin causes a decrease in the level of endogenous antioxidant reserves viz. SOD and CAT and shows an increase in the lipid peroxidation of the heart when compared with the normal. Pretreatment with MEIC 200 and 400 mg/kg showed a significant increase in the SOD (**P<0.01) and CAT (**P<0.01). While significantly decreasing the level of MDA (**P<0.01) in a dose-dependent manner, when compared with the control. The antioxidant tissue markers and lipid peroxidation is shown in Table 5.

Table 5

Antioxidant enzymes and lipid peroxidation of heart tissue homogenate of rats

Histopathological Changes on Dox-induced Cardiotoxicity

Normal rats showed normal cardiac fibers without any damage. The heart sections obtained from Dox-treated animals showed abundant areas of necrosis and aggregation of acute inflammatory cells and damaged vascular spaces. Animals pretreated with MEIC 200 and 400 mg/kg showed improvement in the cell integrity evidenced by absence of necrosis, less vacoulization of the cytoplasm and maintenance of normal integrity of the cardiac muscles [Figure 2].

Figure 2

Histopathological images of heart pretreated with methanolic extract of Ixora coccinea Linn. (MEIC) leaves by doxorubicin-induced cardiac toxicity. a – Normal, b – Control, c – MEIC – 200 mg/kg, d - MEIC – 400 mg/kg

Discussion

Doxorubicin is converted to semiquinone by mitochondrial, lysosomal and cytosolic enzymes. Semiquinone is a charged moiety that readily donates an electron to an oxygen molecule, resulting in generation of an oxygen free radical or superoxide ion/hydroxyl radicals. Due to the presence of less developed antioxidant defence mechanisms of heart, they are particularly vulnerable to apoptosis by anthracycline-induced reactive oxygen species. Therapeutic strategies, designed to augment cellular endogenous defence systems as antioxidants have been identified as a promising approach to combat against Dox toxicity.[19] The present study also evidenced the formation of free radicals which brought about the hemodynamic, biochemical and the histopathological changes in rats treated with doxorubicin.

Doxorubicin-treated animals have shown scruffy fur as well as significantly decreased body weight, heart weight and heart/body weight ratio. The decrease in body weight in this study is in accordance with other studies[20] and it may be attributed to reduced food intake and inhibition of protein synthesis due to Dox treatment compared to normal group. Our study demonstrated a significant increase in the bodyweight gain, heart weight and heart/body weight ratio when compared with the control.

Doxorubicin treatment causes prolongation of P wave, QRS complex, QT interval and RR interval while reduces cardiac cycle.[21] The ST segment elevation was also observed. The consecutive loss of cellular membrane damage due to oxidative stress might be characterized by ST elevation.[22] Treatment with MEIC reflected reduction in P wave, QRS complex, QT interval and RR interval while cardiac cycle was increased, the ST segment was also near to normal. The ECG changes shown by the methanolic extract of I. coccinea leaves may possess protective effect or cell membrane stabilizing action on the myocardium.

As far as hemodynamic study is concerned, Dox caused a decrease in the systolic, diastolic, mean BP and heart rate,[23] this is probable due to effect of Dox on the myofibrils, cause its disruption[24] hence the systolic, diastolic, mean BP and heart rate decreases. Our study demonstrated an increase in the systolic, diastolic, mean BP and the heart rate when compared with the control. This effect confirms the protective effect of the extract on the myocardium.

Deficiency of oxygen supply or glucose supply may cause damage to the myocardial cell membrane leading to permeable and ruptures so that the enzymes leaks out. These enzymes are also called as specific biomarkers, can be estimated to check the damage. Doxorubicin causes an elevation in levels of CK – MB, LDH, SGOT and SGPT when compared with the normal. Treatment with MEIC leaves has shown a significant decrease in the level of these enzymes suggesting the protective or membrane stabilizing effect of the extract on the myocardium.

Oxidative stress and mitochondrial dysfunction are associated with disease and toxic process. It results from over production of ROS, often leading to peroxidation of membrane phospholipids and production of reactive aldehydes.[25] Treatment with doxorubicin cause a decrease in the antioxidant stores of the heart viz., catalase and superoxide dismutase while the extent of lipid peroxidation increases when compared with the normal. Our study demonstrated a significant increase in the endogenous antioxidant stores of CAT and SOD while the MDA levels were decreased when compared with the control. These results indicate the protective effect or free radical scavenging effect of the MEIC in oxidative damage done by doxorubicin. The presence of steroids, tannins, flavonoids, glycosides and alkaloids might be responsible for the free radical scavenging and antioxidant activity of the extract which in returns provide cardioprotection.

Histopathological examination of myocardial tissue obtained from normal animal exhibited clear integrity of myocardial membrane. The heart sections obtained from Dox-treated animals showed disruption of several subcellular elements including loss of myofibrils, swelling of mitochondria, vacuolization of the cytoplasm, formation of lysosomal bodies and dilation of the sarcotubular system.[26] Treatment with the MEIC leaves demonstrated less disruption of the myofibrils and less vacoulization of the cytoplasm. This further confirms the membrane stabilizing effect of the extract.

Conclusions

The present study suggests the cardioprotective effect of methanolic extract of Ixora coccinea Linn. leaves on doxorubicin-induced cardiac myopathy by boosting the endogenous antioxidant stores and blunting the oxidative stress. Further studies focusing on the isolation, characterization and purification of the active constituent and elucidating the exact mechanism of action is to be carried out.