Cardioprotective Effect of Ethanolic Flower Extract of ClitoriaTernatea on Doxorubicin Induced Cardiotoxicity in Rats

Tamizhselvi A*1, Durairaj S2, Mohamed Sadiq A3, Gopinath G1, Rajeshkumar S4 1Department of Bio-Chemistry, Indo-American College, Perungalathur 604407, Cheyyar, Tamil Nadu, India 2Department of Zoology, Aringnar Anna Government Arts College, 604407, Cheyyar, Tamil Nadu, India 3Department of Bio-Chemistry, Adhiprasakthi College of Arts &Science, G.B Nagar, Kalavai632506, Tamil Nadu, India 4 Department of Pharmacology, Nanobiomedicine Lab, Saveetha Dental college, SIMATS, Chennai, Tamil Nadu, India


INTRODUCTION
Doxorubicin-an anti-cancer candidate drug, which furthermore provoke myocardial damage in addition to that it is generally used as a chemotherapeutic mediator for a broad spectrum of hard cancer and malignancies, Doxorubicin having ironanthracycline complex produces oxidative stress, which in turn, causes ruthless injure to the membrane layer, and hinder with the morphology of cytoskeleton (Billingham et al., 1977). The ironanthracycline complex provoke oxidative stress and produce free radicals by doxorubicin (van Acker et al., 1996) which aim the heart tissue to lipid peroxidation cause permanentdamage to myo ibrils, cytoplasmicdialation with sarcoplasmic reticulumvacuolization, increased quantity of lysosomes, myocyte necrosis also in lammation of mitochondria, (Minotti et al., 2004), biosynthesis of protein and nucleic acid also hindered (Monti et al., 1995), adrenergic alteration,Na+K+ATPase also shrink (Geetha and Devi, 1992), modi ication in calcium transfer of sarcoplasmic along with the difference in electrolytes balance of heart musclesof doxorubicin induction (Siveski-Iliskovic et al., 1994). The cell transformed doxorubicin into semiquinone by enzymes present in mitochondrial and lysosomal. The electric Semiquinone voluntarily contributes oxygen to an electron, resulting in the making of oxidative stress to cause free radicals liberation. Superoxide dismutase catalyzed the con iguration of hydrogen hydroxide from Superoxide (Jain, 2000).CVD is a group of disorders or diseases of the heart and blood vessels, which also include atherothrombotic diseases such as myocardial and cerebral infarction commonly called heart attack and stroke in that order. The events like atherosclerotic plaque creation, endothelial dysfunction (Virmani et al., 2000). Atherosclerosis is an in lammatory disease of the large arteries describeatherosclerotic plaques formation. The possible foundation of endothelial dysfunction leading to atherosclerosis contains enhanced free radicals, elevated and modi ied low mass lipoproteincholesterol, hypertension, diabetes mellitus, genetic alterations and prominent plasma homocysteine absorption (Singh et al., 2002). In the majority of cases, atherosclerosis-related clinical events, such as MI or ischemic stroke, are caused by the burst of a vulnerable atherosclerotic lesion (Badimon et al., 2012). Butter ly pea (Clitoriaternatea Linn) is used as folk medication. The family tree and germ are use as the stress tonic and laxative. Flowers of butter ly pea restrains anthocyanins. The red violetblue color of plant and lower is because of Anthocyanins (Harborne, 1998). (A2, A1,B2,B1,D2 and D1) are the main anthocyaninsternatins (Abernethy, 1960). The diversity of shades of the lower is owed to a very small digit of a different tincture. These tincture contain the matching carbon skeleton, with diverse only in the character of the substituent grouping (Abernethy, 1960) . The current job be accept to revision the cardioprotective role of EECT on cardiac toxicity induced by doxorubicin.

Plant
Clitoraternatea lowers be purchased from koyambedumarket, Chennai, Tamilnadu and were authenticated Dr.V.Gangadevi, Assistant Professor, Botany Department, Aringnar Anna Government Arts College, Cheyyar, Tamilnadu. The herbarium have be present on behalf of auxiliary indication. The lower were shade dried and ine pulverized using pulverize and maintained in an airtight container at 4º C and used for further study.

Preparation of extract
The inely powdered dried lowers of clitoraternatea convert to crude powder. The crude powder was extracted with solvent ethanol through soxhlet until them colorless. Then it was pressure evaporated by rotary evaporator below 45ºC wait the formation of syrupy of the extract and transferred it in a clean dish at room temperature.17.3% w/v was the yield originated.

Animals and chemicals
Thirty animals of either sex Wister albino rats through an common weight of 221.54 ± 2.56 gms taken for study acquiredinBiochemistry Department animal House of Adhiparasakthi College of Arts And Science .G.B.Nagar, Kalavai, Vellore-Dist,Tamilnadu. Normal food with libitum water was given to the experimental rats for the entire period of period. Animal husbandry, tests and toxicology were carried out with doctrine guideline regularly. Prior to experiments, animals were familiarized for two days to adopt the laboratory condition to minimize unwanted stress. The animals are sustained under normal laboratory conditions of 25± 1ºC through a 12 h light-dark cycle. Care was full to evade any sort of tense provision. All experimental procedure was executed between 9 and 11 a.m. The entire experiments done with proper obtained from Institutional Animal Ethical Committee, Adhiparasakthi College of Arts And Science, G.B.Nagar, Kalavai, Tamilnadu (IEACAP-CAS/01/2017/02) Doxorubicin (Fresenius Kabi) was purchased from local market. The reagents, Glasswares and water used in this reading were of systematic score and standard.

Safety evaluation / Toxicity studies
Acute oral toxicity readings in taken animals were approved with guidelines of OECD-423. Five doses (50, 300, 500, 1000, 2000mg /kg body weight)    of ethanol extract of clitoraternatea were vocally directed to groups of similar age and weight. The taken rats were supervised for 60 minutes continuously and after that hourly for 4 hour and inally for every 24 hours until the inal protocol for some warning sign of toxicity and transience (Chandan et al., 2007). The indication of embarrassment to the animals were originate at a dosage of the 2000mg/kg bw EECT. Therefore the dose of 1000mg/kg bwup to 1,000 mg/kg was chosen to be safe also the dose of 100, 200 & 300 mg/kgbw of EECT taken to study the cardioprotective commotion.

Experimental Protocol
The experimental protocol applies for assessment of cardioprotective effect of ethanolic extract clitoraternateaagainst DOX-induced cardiotoxicity is as follows. The rats were erratically allocated into six groups of ive each.
Group I: Control group received distilled water for 14 days.
Group II: DOX-induced group received a single dose of DOX 15mg/kg I.P on 1 st day only.
GroupIII: Standard Drug treated group received a single dose of DOX 15mg/kg I.P on 1 st day, Metoprolol (10 mg/kg bw) for the other 13 days.
GroupV: Treated Group2 received DOX 15mg/kg I.P on the 1 st day and 200mgEECT for other 13 days.
GroupVI: Treated Group3 received DOX 15mg/kg I.P on the 1 st day and 300mgEECT for other 13 days.
At the end of the 14 th day, the animals were anaesthetized Ketamine -Xylazine and then sacri iced by a high dose of ketamine. Blood samples were collected immediately for enzyme assays. The serum was prepared by centrifuging the blood samples at 3000 rpm for 5 min. The heart of the rats were rapidly dissected and washed in isotonic saline and homogenized quickly with ice-cold 0.1M Tris HCl buffer (pH-7.5) and stored.

Cardiac biochemical markers
The LDH, AST and CK-MB actions were dogged from serum according to normal technique using standard kits.

Lipid peroxide content
Lipid peroxide enzyme levels was determined by thiobarituricacid (TBA) reaction with Malondialdehyde (MDA), a creation create dowing to the peroxidation of lipid membranes (Ohkawa et al., 1979).

Catalase activity
The CAT activity of cardiac tissue was resolute according to the method described by Aebi (Aebi, 1984) the method based on the determination of the H 2 O 2 decomposition rate at 240 nm. The values are expressed as U/mg protein.

Glutathione content
Reduced Glutathione was estimated by the method of Ellma (Ellman, 1959) (Ellman GLet al., 1959) About 1.0 ml of 10% TCA was added to 1.0 ml of homogenate and centrifuged. The supernatant was treated with 0.5 ml of Ellmans reagent and 3 ml of phosphate buffer (pH-8.0). The color developed was measured at 412 nm.

Superoxide dismutase activity
The SOD was determined by the ability of the enzyme to inhibit the oxidation of adrenaline to adrenochrome (Saggu et al., 1989). The 0.05 ml supernatant was added to 2.0 ml of carbonate buffer as well as 0.5 ml of 0.01MEDTA solution. 0.5 ml of epinephrine was added for the initiation of reaction. The auto-oxidation of adreno chrome was measured by following change in OD at 480 nm. The results are expressed at U/mg protein.

Assay of total protein
To0.3 ml of solution of tissue homogenate to that alkaline copper sulphate, 2ml kept for 10 min at RT then 02. Ml for folinciacalteau added and kept for 30 min incubation and read at 660nm with standard BSA.The assay of total protein carried out by the (Lowry et al., 1951).

Histopathology of Heart tissue
On the 15 th Day,the heart is cut off, clean with isotonic saline also homogenized among 1 M Tris Hcl buffer. Followed via the tissues be presented in 10% buffer unbiased formalin solution. Following complex tissues be ixed in paraf in -wax and segments be hacked and stain by hematoxylin and eosin. The stained tissue coated slides were examined with microscopy.

Statistics
The collected data were articulated as the mean ± SD for analysis of statistical data, group were balanced by ANOVA, tag alonged by Dunnett's test. The p values < 0.05 was measured considerable.
In DOX-induced group, the mammal's hair change into muddled and yellowish to ruddy dark development is also seen. The Animals have dark exudates- roughly the eyeball along with nudge, elastic luid feces, the existence of peritoneal watery, necrosis at the location of insertion. Animals are in pitiable health, slow-moving and swelling of liver and stomach that distinguish among the control animals. The above annotations be noticeably compacted in the group treated with EECT (200 &300 mg /kg bw) when contrasted with DOX-induced animals group.
In Doxorubicin-induced rats, there is a reduce the mass of the body, heart tissue and ratio of their weights on inal day (Figures 1 and 2).EECT (200 mg/Kg and 300 mg /Kg ) established major boost mass of body, heart and its ratio (p<0.05 ) when contrast with DOX-induced group (Table 1).
The protein content in the DOX-induced rats demonstrated a decrease in the protein levels when compared to control. Animals treated at a dose of 200 7 300 mg/kg bw of EECT illustrate a considerable high in the levels of protein in contrast to DOX-induced group.

Histopathological Studies
Histopathological assessments of heart tissue achieved as standard control animals established an understandable stead fastness of myocardial covering. Standard control animals demonstrated standard cardiac iberssmart of which even harmed. Cardiac muscle section ofDOX-induced ratsdemonstrate suf icient region of necrosis with aggregations,cells with severe in lammation and vascular spaces also injured. Rats treated with EECT of 200 & 300mg/kg bwshown an enlargement of the cells reliability established by be short of necrosis, in lammatory cells are noticeable diminish in size and protect the usual morphology of the heart tissue( Figure 6).
The ethanolic lower extract of ClitoriaTernateahave exposed momentous cardio protection through Doxorubicin-induceded cardiac toxicity. Single admin of Doxorubicin(15mg/kg bw) will demonstrate as a source of cardio toxicity in rats (Somashekar, 2015). In this experiment, we developed a Cardiotoxic rat model by injecting a single dose of 15mg/kg bw of DOX and it has been shown to as signi icantly diminished body, heart and ratio of their masses. The diminish in the mass of the body in this study is in agreement by means of additional studies (Herman et al., 2000)and this is because of food eaten by the animals and it may diminish protein production due to Doxorubicin. The ethanolic extract will demonstrate a ampli ication in the mass of the body, heart and ratio.
DOX induction created a considerable increase of Creatinine kinase (Somashekar, 2015), LDH (Garba and G, 2005). An elevated level of this oxidative enzymes will back to normal by EECT treatment signi icantly. This mitochondrial dysfunction and oxidative stress is because of the DOX induction causing sick to the rats. DOX will cause membrane lipid peroxidation, production of ROS and also the reactive aldehyde. (Morin et al., 2001).SOD and CAT enzymes shown a signi icant decrease but an increase in LPO on DOX-induced group when compare to the Normal control group. Treating EECT on DOX-induced group considerably get the level of these enzymes back to normal. This provides evidence that the avoidance of oxidative injure by EECT. The cardiac tissue Histopathology observation of the groups shown a signi icant changes in EECT treated when compared to DOX-induced group. The heart sections acquired since Doxorubicin-induced animals demonstrated commotion of numerous subcellular feature which includes myo ibrils loss ,mitochondria swelling, cytoplasmic vacuolization, sarcotuburdialation and lysosomal formation (Olson et al., 1974).The architecture of the heart close to normal with control group while treating with 200mg/kg and 300mg/kg bw of EECT. The presence of phytochemical constituents like Triterpenoids, Flavonoids, Tannins, Soponins, glycosides, sterols, carbohydrates, alkaloids, proteins and carbohydrates will strength the cardio protectively of EECT in DOX-induced Cardiotoxicity of the taken experimental animals.

CONCLUSIONS
EECT demonstrated a persuasive anti-cardiotoxic outcome in the taken experimental animal by DOX persuaded cardiotoxicity / myocardial necrosis. Treatment with EECT may effect LDH, CK-MB and AST activity. This put forward that EECT might be the reason in the reduction and avoidance of the severe cardiac complications encountered by Doxorubicin. The EECT extract is a novel strategy with the aim for defending against Doxorubicininduceded cardiotoxicity. In conclusion the over statistics put forward to facilitate EECT has the potential antioxidant movement in avoiding the cardiotoxic possesses on persuade by Doxorubicin.