INTRODUCTION:

At present cardiovascular diseases (CVD’s) are being the major health burden with superlative mortality rate globally. The one fourth of all mortality is accountable to myocardial disorders¹. Myocardial Ischemia and Cardiopulmonary arrest been the prevalent causes, these are also extremely responsible for more than 85% of CVD deaths.

As indicated by the Global Burden of the Disease Study age-institutionalized evaluations (2011), almost a quarter (24.8 per cent) of all passing in India are owing to cardiovascular diseases².

Several Chronic diseases including CVD are result of Oxidative stress that develops due to increased origination of free radicals and ROS in the body³. Trend growing in researches for evaluation of medicinal plants for CVD by using cardio toxic models for example, doxorubicin induced cardiotoxicity to investigate cardioprotective properties of many medicinal plants.

Cardiotoxicity (C.T) is referred as any toxic effect on heart muscles, although the exact definition of it is still lacking⁴. C.T caused by chemotherapeutic agents can be toxic in nature cause myocardial damage and known to have high prevalence⁵. It develops irregular heart rhythms as the heart becomes incapable for pumping required blood to distribute essential O₂ and nutrients to the whole body. C.T leads to cardiopulmonary arrest, myocardial ischemia/ infraction, QT prolongation, left ventricular failure, pericarditis, thromboembolism and exaggerated blood pressures⁶.

Doxorubicin (DOX) an anthracycline drug and potent broad spectrum chemotherapy agent, useful in several kinds of cancer like leukemia, strong tumors, breast cancer, small cell lung carcinoma and esophageal carcinoma. Many studies demonstrated administration of DOX above supra maximal dose causes cardiac dysfunctions due to generation of excessive reactive oxygen species (ROS)⁷. The most important cellular damage caused by ROS includes lipid peroxidase, crosslinking of protein, DNA damage^8,9.These leads to cardiomyopathy damaged mitochondrial biogenesis, decreased Na⁺/k⁺ ATPase activity, overloading of calcium¹⁰.

Plant based antioxidants which can protect the cells from oxidative damage should be incorporated in the potential antioxidant therapy. Therefore there is a need for discovery of alternative, natural, safer sources of antioxidants.

Tamarindus indica linn is a part of the Fabaceae family, commonly referred to as tamarind widely used as traditional food and in ayurvedic herbal therapies. Several studies reported T. indica is a multipurpose tree, every part of which finds at least some use either nutritional or medicinal. The pericarp of tamarind fruit contains phenolic antioxidants¹¹.Its polyphenolic profile is dominated by proanthocyanidins. These proanthocyanidins are a type of oligomeric flavanoids also referred as condensed tannins mostly found in peels of fruits and vegetables are utilized as powerful antioxidants¹².

Thus, keeping in view the above mentioned facts, the current study was designed to evaluate the cardioprotective activity of Tamarindus indica pericarpic extract against doxorubicin induced cardiaotoxicity in albino rats.

MATERIALS AND METHODS:

Drugs and chemicals:

Doxorubicin Hydrochloride injection (Doxoruba IP-50mg/25ml; Company: Getwell Oncology Pvt. Ltd) was purchased from local hospital pharmacy. Kits for serum analysis were procured from coral diagnostics (LDH, CK-MB, AST, ALT and Calcium).

Collection and Authentication of plant material:

The pericarps (shells) of Tamarindus indica fruits were collected from local market of Hyderabad, Telangana, India. The plant material was authenticated by Dr. P. V. Prasanna Scientist F at Botanical Survey of India, Deccan Regional Centre Hyderabad. Voucher specimens were stored in the department of Pharmacology, CMR College of Pharmacy, Hyderabad, Telangana state, India.

Preparation of the extract:

The pericarps (shells) of tamarind fruits were peeled, washed with distilled water and dried under sunshade, and then they were powdered using a mechanical grinder. Then the powdered material was mixed with concentration of 80:20 respectively (Water: Ethanol) and kept at 25^oC for one week with occasional shaking¹³. Thereafter, it was stirred for 20min and filtered. The filtrate was dried in rotary evaporator (ROTA VAP). The hydroalcoholic extract of Tamarind pericarp (HAETP) was subjected to preliminary phytochemical investigation¹⁴ and stored in refrigerator at 4^oC for further studies.

Animals:

Adult male albino rats of Wistar strain weighing 170-200g were obtained from Sainath Agencies, #1-6-197/45/D, Balaji Nagar, Musheerabad, Hyderabad-500048, (282/PO/Bt/S/2000/CPCSEA). Acclimatization of animals to the laboratory environment was done for a week. The animals were randomly housed in polypropylene cages along with paddy husk as bedding, in a well ventilated room under hygienic conditions and were exposure to 12 hour day and night cycle. The animals were fed with commercial rat pellet feed and were given water ad libitum. All the experimental methodology and conventions utilized in this investigation were approved by the Institutional Animal Ethics Committee (IAEC) of CMR College of pharmacy, Hyderabad, (1657/PO/Re/S/12/CPCSEA).

Groups:

The adult male rats of wistar strain has been divided into 5 groups of 6 animals each and exposed to following treatments for 30 days.

Group I: Normal control group: received normal saline 5 ml/kg body weight p.o as vehicle for 30 days.

Group II: Disease control group: Treated with single dose of DOX-20mg/kg body weight i.p on 29^thday.

Group III: Standard control group: Received Amlodipine 5 mg/kg body weight p.o for 30 days and DOX-20mg/kg body weight i.p on 29^thday.

Group IV: HAETP 200mg: Received HAETP 200mg mg/kg body weight p.o for 30 days and DOX-20mg/kg body weight i.p on 29^thday.

Group IV: HAETP 400mg: Received HAETP 400mg mg/kg body weight p.o for 30 days and DOX-20mg/kg body weight i.p on 29^thday.

After 31^stday, blood samples were collected from the retro orbital plexus for the estimation of serum analysis. Later, animals were sacrificed under anesthesia and hearts were isolated for in vivo antioxidant studies, membrane bound enzyme assays and histopathological studies.

Acute toxicity study:

Acute toxicity study was performed according to the guidelines of OECD 425. Two groups of male Wistar rats (n=3 in each group) were used for the study. First group received HAETP-2000 mg/kg p.o, second group received vehicle, distilled water. After administration, animals were observed individually at least once every 30min, periodically during the first 24 h, with special attention given during the first 4 hours and daily. Thereafter, observed continuously for a total of 14 days for the profiles such as lethargy, alertness, irritability, spontaneous activity, changes in skin, fur and eyes, behavior pattern, tremors, convulsions, salivation, defecation and urination, coma and death. HAETP was well tolerated and showed no morbidity and mortality. Based on the results, the 1/10^th and 1/5^thof the maximum tolerated dose were selected i.e. 200 mg/kg and 400mg/kg respectively by oral route for further studies.

Dose selection and experimental induction of myocardial toxicity or cardiac stress:

Dose of Doxorubicin as 20mg/kg body weight i.p was selected based on previous studies¹⁵. Commercially available doxorubicin was used for experimental induction of myocardial toxicity or cardiac stress.

Estimation of biochemical parameters:

Serum markers:

At the end of the study, blood samples were collected from the retro orbital plexus. Blood samples were kept aside for approximately 1hr at room temperature and centrifuged at 2500rpm at 4^oC for 15min to separate the serum from blood. The serum samples were used for estimation of various biochemical parameters such as Lactate dehydrogenase (LDH), Aspartate transaminase (AST), Alanine transaminase (ALT), Creatine kinase (CK-MB) and Calcium by using coral kits and semi auto analyzer (Inkarp ES-100).

Biomarkers of the oxidative stress:

After blood collection, animals were sacrificed by cervical dislocation and were cut open to isolate heart and were weighed immediately. The isolated heart was chopped into slices and was chilled in the ice cold potassium chloride (1.15%). These fine slices were homogenized in ice cold potassium chloride (1.15%) using tissue homogenizer. It was then centrifuged at 5000rpm at 4^oC for 20min to separate the homogenate from cellular debris. The supernatant was collected and used to estimate oxidative stress markers such as Malondialdehyde level as indicator of Lipid peroxidation (LPO)¹⁶, Reduced glutathione (GSH)¹⁷ and Catalase (CAT)¹⁸.

Membrane bound enzymes:

The above prepared tissue homogenates were also used for estimation of membrane bounded enzymes such as Na⁺/k⁺ ATPase¹⁹, Ca⁺⁺ ATPase²⁰ and Mg⁺⁺ ATPase²¹ activities.

Histopathological studies:

The parts of the hearts after euthanasia were stored in 10% (w/v) buffered formalin and embedded in paraffin, sections were cut at four µm thick, stained with Hematoxylin and Eosin (H and E) reagents. These sections were observed under light microscope for histopathological changes.

Statistical analysis:

The values were expressed as Mean ±SEM. The Statistical analysis was carried out by one-way Analysis of variance followed by post hoc Dunnett’s test using graph pad prism 5.0. The values are significant at p<0.05.

RESULTS:

Preliminary phytochemical screening:

Preliminary phyto chemical studies of hydro alcoholic extract of Tamarindus indica. pericarp specified in the Table.1

Table 1: List of phytochemical constituents present in HAETP

Name of the phytoconstituent	HAETP
Alkaloids	Present
Saponins	Present
Carbohydrates	Present
Flavonoids	Present
Glycosides	Present
Proteins	Absent
Steroids	Present
Tannins and phenolic compounds	Present
Amino acids	Present

Effect of HAETP treatment individually on body weights on 1^st and 31^st Day:

Body weight in disease control group showed significant decrease compared to normal, whereas in standard and HAETP treatment groups there was a significant increase in the body weight as shown in Table.2.

Effect of HAETP treatment on heart weight on 31^stDay:

The disease control group showed significant increase in heart weight with comparison to normal and standard group. The treatment groups standard amlodipine, HAETP 200mg/kg and HAETP 400mg/kg showed significant decrease in heart weight compared to the disease control group as shown in the Table.3.

Table 4: Effect of HAETP treatment on serum cardiac cardioprotective parameters

S. No	Name of the group	LDH (IU/L)	AST (mg/ml)	ALT (mg/ml)	CK-MB (IU/L)	CALCIUM (mg/dl)
1	Normal	340±5.61	42.3±1.98	34.3±1.09	334±11.4	5.88±0.169
2	Disease control (Doxorubicin 20mg/kg)	553±18.3^d	97.6±1.65^d	77.5±2.03^d	637±19.5^d	18.1±0.163^d
3	Standard Amlodipine (5 mg/kg)	356±11.6^a	44.8±0.909^a	35.1±1.13^a	337±12.0^a	4.92±0.233^a
4	HAETP200mg	473±2.79^ad	62.9±3.58^ad	47.0±1.45^ae	503±26.4^ad	8.24±0.713^ad
5	HAETP400mg	420±18.0^ae	51.6±1.53^a	42.7±0.295^af	451±6.08^ad	6.13±0.281^a

Values are represented as Mean ± SEM. Statistical analysis performed using one way ANOVA followed by post hoc Dunnett’s test. ^ap<0.001, ^bp<0.01 and ^cp<0.05 Vs Disease control; ^dp<0.001, ^ep<0.01 and ^fp<0.05 Vs Standard Amlodipine.

Effect of HAETP treatment on oxidative stress markers against doxorubicin induced cardiotoxicity:

Effect of HAETP on oxidative stress markers such as lipid peroxidation, reduced glutathione and catalase of cardiac tissue homogenate was summarized in Table.5. There was significant increase in the lipid peroxidation level and decrease in the levels of reduced glutathione and catalase observed in disease control (p< 0.001) when compared to the normal and standard groups. Significant improvement was observed in the levels of lipid peroxidation, catalase and reduced glutathione (p< 0.001) in standard Amlodipine group when compared to the disease control group. Treatment with HAETP showed significant recovery from lipid peroxidation and increased levels of reduced glutathione and catalase in a dose dependent manner (p< 0.001 and p< 0.01) when compared to the disease control.

Table 5: Effect of HAETP treatment on oxidative stress markers

S. No	Name of the group	LPO (µM/mg)	GSH (µM/mg)	CAT (µM/mg)
1	Normal	3.44±0.073	8.62±0.425	0.593±0.04
2	Disease control (Doxorubicin 20 mg/kg)	11.1±0.301^d	2.23±0.26^d	0.074±0.01^d
3	Standard Amlodipine (5 mg/kg)	4.11±0.23^a	8.61±0.26^a	0.632±0.02^a
4	HAETP 200 mg	6.61±0.14^ad	6.05±0.49^b	0.484±0.02^ae
5	HAETP 400 mg	4.80±0.27^a	8.40±1.53^a	0.544±0.02^a

Effect of HAETP treatment on membrane bounded enzymes of heart against doxorubicin induced cardiotoxicity:

Effect of HAETP on membrane bound enzymes such as Na⁺/K⁺-ATPase and Mg⁺⁺-ATPase and Ca⁺⁺-ATPase of cardiac tissue homogenate was summarized in Table.6. There was significant decrease in the Na⁺/K⁺-ATPase and Mg⁺⁺-ATPase levels and increase in the levels of Ca ⁺⁺-ATPase observed in disease control (p< 0.001) when compared to the normal and standard groups. Standard Amlodipine group showed significant increase (p< 0.001) in the Na⁺/K⁺-ATPase and Mg⁺⁺-ATPase levels and decrease (p< 0.001) in the levels of Ca⁺⁺-ATPase levels when compared to the disease control group. Treatment with HAETP showed significant recovery from Ca⁺⁺-ATPase and increased levels of Na⁺/K⁺-ATPase and Mg⁺⁺-ATPase in a dose dependent manner (p< 0.001), (p< 0.01) and (p< 0.05) when compared to the disease control group.

Table 6: Effect of HAETP treatment on membrane bounded enzymes

S. No	Name of the group	Na⁺/k⁺ ATPase (µM/mg)	Ca⁺⁺ ATPase (µM/mg)	Mg⁺⁺ ATPase (µM/mg)
1	Normal	1.85±0.04	0.688 ± 0.06	0.0638 ± 0.002
2	Disease control (Doxorubicin 20 mg/kg)	1.24±0.04^d	1.60 ± 0.05^d	0.0312 ± 0.002^d
3	Standard Amlodipine (5 mg/kg)	1.83±0.03^a	0.698 ± 0.06^a	0.0608 ± 0.003^a
4	HAETP 200 mg	1.65±0.10^a	1.32 ± 0.016^bd	0.0458 ± 0.007
5	HAETP 400 mg	1.69±0.10^a	0.783 ± 0.07^a	0.0497 ± 0.007^c

Histopathological studies:

Histopathological examination of treatment groups are shown in the Fig.1 (a-e). Normal control rat showed normal integrity of myocardial fibres with no vacuoles, necrosis, inflammation and infarction. The heart section of disease control rats showed largely covered irregular shaped hypertrophic fibres with loss of striations, intracytoplasmic vacuoles present in fibres, leukocyte infiltration and inflammation of myocardium. Standard Amlodipine (5mg/kg) treated rats showed normal shaped sized myocardial fibres with no vacuolation. HAETP 200mg/kg and 400mg/kg pretreated rats showed mild to moderate protection against doxorubicin toxicity evidenced by reduction in the loss of striations, necrotic region, infiltration of Leukocytes, and increase in restoration of normal integrity of myocardial fibres.

Fig. No. 1 (a-e). Effect of HAETP treatment on histopathological studies against doxorubicin induced cardiotoxicity.

DISCUSSION:

The present study was done to investigate cardioprotective role of HAETP against DOX induced cardiotoxicity. Our experimental data suggests that HAETP prevents DOX induced cardiotoxicity in rats. Following lines are emphasized as evidences of the present study. The preliminary phyto-chemical evaluation was performed with HAETP and it revealed that it contains flavanoids, phenols, tannins, alkaloids, glycosides as major components²². In DOX treated or disease control group, development of color change of animal fur to pink has been seen in post observational days was follow on with red exudates at eyes and nose. These were less seen in the HAETP pretreated animals, revealing its protective property.

The DOX treated group showed increase in heart weight indicating loss in myofibrils, dilating sarcoplasmic reticulum, and enlargement of mitochondria. These changes were opposed in the HAETP pretreated groups. The HAETP pretreated rats shown significant increase in body weights contrast to DOX treated group this might be due to fewer intakes of water and food. Increase in body weight in the HAETP pretreated rats indicate that they have ability to alter the oxidative stress occurring from doxorubicin treatment

The LDH level rises in myocardial infarction and it raises 4-5 times more to normal also up to 12 times the value of normal. CK-activity gets elevated in cellular damage contributing skeletal muscle, heart muscle, brain injury etc. Increase in levels of certain CK-isoenzyme in blood is of particular diagnostic value. The LDH and CK-MB values are important aspects of measurement of late and early cardiac injuries²³.

Serum AST and ALT enzymes are very important for the smooth regulation in breaking down and making up processes in human bodies²⁴. The current study reports that AST, ALT proportions were increased in the disease control group indicating inflammation and necrosis.

The increase in levels of LDH and CK-MB in DOX treated group reveals damage particularly occurred in cardiac tissues and indicates myocardial infarction. Pretreatment with HAETP-200mg/kg showed slight reduction and HAETP-400mg/kg showed significant decrease in the proportions of DOX -induced elevated LDH, AST, ALT and CK-MB indicating moderate protection of myocardial tissues towards doxorubicin toxicity.

Doxorubicin induced cardiotoxicity shows rise in intracellular calcium concentration in the heart tissues due to impairment of calcium handling which prompts alterations in contractions and relaxations of the heart²⁵. It also suggests that it causes interferences in mitochondrial calcium regulation and irreversible decrease in loading capacity leading to loss in myocardial function²⁶. This was opposed in HAETP pretreated (200mg/kg and 400mg/kg) groups contrast to DOX treated group.

The appropriate mechanism responsible for cardiotoxicity which is instigated by doxorubicin has not been clearly revealed from the present study but due to lot of evidences from earlier studies it shows doxorubicin suppresses characteristic antioxidants and raises oxygen free radicals prompting high levels oxidative stress promoting alterations in cardiac cells and typically cardiac injury²⁷. In present study, DOX treated group showed significant rise in MDA concentration in heart muscles prompting high range of lipid peroxidation, also GSH and CAT levels were suppressed indicating myocardium damage.

HAETP pretreated rats showed dose dependent reduction in cardiac MDA concentration additionally rise in GSH and CAT contrast to disease control group which reveals that it has a protective role towards cardiac tissues.

Heart function gets affected by change in the activities of ion pumps namely, Na⁺/k⁺ ATPase, Ca⁺⁺ ATPase and Mg⁺⁺ ATPase. These membrane bound enzymes have important role in contraction and relaxation properties of cardiac muscles. In this study DOX treated group showed decreased levels of Na⁺/k⁺ ATPase, Mg⁺⁺ ATPase and increase in Ca⁺⁺ ATPase level evident that doxorubicin destroys cell membranes as reported in earlier studies²⁸. HAETP pretreated (200mg/kg and 400mg/kg) groups restored the levels of Na⁺/k⁺ ATPase, Ca⁺⁺ ATPase and Mg⁺⁺ ATPase indicating membrane stabilizing properties which support the previous studies.

Histopathological changes of cardiotoxicity induced by DOX includes increasing order of severity, swelling of sarcoplasmic reticulum, vacuolization in cytoplasm, degeneration of myocardial fibres, disruption in myocytes and fibrosis²⁹. In our study we observed no morphological changes in normal control rats and heart showed normal uniform size, shape and arrangement of myocardial fibres with no infiltration and necrosis. In heart sections of DOX treated group, hypertrophic fibres, vacuoles in the cytoplasm, alteration in the myocyte structure, damaged microtubules, infiltered inflammatory cells, degeneration of myocardial fibres were observed which were in line with previous reports. These findings were reversed in HAETP pretreated groups and standard amlodipine group. Less degeneration of myocardial fibres and only single vacuole was observed in the HAETP 200mg/kg. In HAETP 400mg/kg group and standard amlodipine group normal configuration of cardiac muscle fibres with normal shaped and sized myocytes were observed which is due to reversal of infiltration of inflammatory components and fragmentation of cardiac fibres. The protection exhibited by HAETP as observed from histopathological alterations may be due to its antioxidant potency to counteract the free radicals.

CONCLUSION:

The present study suggests that HAETP has moderate protective effects towards doxorubicin induced cardiotoxicity in rats. Pretreatment of HAETP (200mg/kg) and HAETP (400mg/kg) showed significant reduction in DOX-elevated serum marker enzymes- LDH, AST, ALT, CK-MB, and Calcium levels. HAETP treated groups showed significant decrease in heart MDA levels with increase in levels of GSH and CAT contrast to DOX treated group suggesting existence of antioxidant properties. Pretreated HAETP groups showed restoration of membrane bound enzymes such as Na⁺ /K⁺ ATPase, Ca⁺⁺ ATPase, Mg⁺⁺ ATPase indicating membrane stabilizing properties. The histopathological studies reveals morphological changes in heart caused by DOX induced cardiotoxicity were reversed by HAETP pretreated groups indicating protective effects against free radicals. However, futher studies should be performed to elucidate detailed cardioprotective mechanism.

ACKNOWLEDGEMENT:

We are thankful to the Principal, and Management CMR College of Pharmacy for providing infrastructure facilities for the present study.

CONFLICT OF INTEREST:

There are no conflicts of interest.

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Received on 04.09.2019 Modified on 10.11.2019

Research J. Pharm. and Tech. 2020; 13(7): 3267-3273.

DOI: 10.5958/0974-360X.2020.00579.X

Groups	Normal control	Disease Control (Doxorubicin 20 mg/kg)	Standard Amlodipine(5mg/kg)	HAETP (200mg/kg)	HAETP (400mg/kg)
Groups	Change of body weights (grams)
1^stday	182±3.07	177±3.33	182±3.07	178±4.01	183±5.58
31^stday	203±4.22	183±2.11	217±3.33	192±1.67	215±9.57

S. No	Name of the group	Heart weights (gm)
1	Normal	0.458±0.00543
2	Disease control (Doxorubicin 20 mg/kg)	0.567±0.0154
3	Standard Amlodipine (5mg/kg)	0.477±0.00715
4	HAETP 200 mg	0.520±0.00447
5	HAETP 400 mg	0.503±0.00667