Cardiovascular diseases (CVDs) are the leading cause of death globally, taking an estimated 17.9 million lives each year. CVDs are a group of disorders of the heart and blood vessels and include coronary heart disease, cerebrovascular disease, heart failure, cardiomyopathy etc.¹ The death rates due to cardiomyopathies are 6 per 1,00,000 population and disability adjusted life years are 166 per 1,00,000 population.² Incidence of death due to drug related cardiomyopathy is 2 % with 10 % decline in left ventricular ejection fraction.³

The use of herbal plants is increasing as protective agents against a number of cardiovascular diseases. By eliminating the generation of free radicals, phytochemicals from natural sources have decreased the risks of heart disease and gained fundamental importance in modern drug systems. Cardio-protection includes “all mechanisms and means that contribute to the preservation of the heart by reducing or even preventing myocardial damage”.⁴

Herbal plants like Tulsi (Ocimum sanctum) and Arjuna (Terminalia arjuna) have shown cardioprotective potential.⁵ Hibiscus rosa sinensis (Malvaceae family) has been found to have antiulcer, antidiabetic, anticonvulsant, anti-inflammatory, antipyretic, antibacterial activity as well as cardioprotective activity.⁶

Since not much literature is available on cardioprotective potential of Hibiscus rosa sinensis, hence, the present study was undertaken to investigate the cardioprotective potential of aqueous extracts of Hibiscus rosa sinensis.

MATERIALS AND METHODS

STUDY DESIGN

The present study was conducted in the Department of Pharmacology, L.L.R.M. Medical College, Meerut for evaluation of cardioprotective potential of aqueous extract of Hibiscus rosa sinensis against Doxorubicin induced cardiotoxicity in albino rats.

After obtaining approval from the Institutional Animal Ethics Committee (registration no.819/GO/ReRc BiBt/S/04/CCSEA), albino rats weighing 150-200gm was procured and maintained under standard laboratory conditions (alternating periods of light and darkness of 12h each and at 25^OC), with free access to standard rat pellet diet and tap water ad libitum. Pregnant female rats were not included in the study. All animal experiments were carried out in accordance of the Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India. As per OECD guidelines, doses of aqueous extract of Hibiscus rosa sinensis to be used in the study were calculated on the basis of previously documented LD50 on rats (OECD-423).

Method of preparation of extract:

Aqueous extract of Hibiscus rosa sinensis:

           Dried Hibiscus rosa sinensis flowers was procured from market in pulvurised form and incubated with 10 volumes (w/v) of boiling water for 20 min and cooled to room temperature. This 10% solution was then clarified by centrifugation, sterilized by filtration through 0.2 micron filters (millipore), frozen, and lyophilized to dryness. This dried extract was then  suspended in water to a final concentration of 50 mg/ml.⁷

STUDY OUTLINE:

Evaluation of cardioprotective activity:

The study was conducted on albino rats weighing 150-250 gm. During the study period of 21 days, 30 rats were randomized into five groups of 6 rats each.

• Group 1 – This group was given Normal saline 1ml/kg orally (control) for 21 days.
• Group 2 – This group in addition to normal saline was administered Doxorubicin (20mg/kg intraperitoneally) on 21^st day of study.⁸
• Group 3 – This group was treated with Carvedilol 30 mg/kg orally for 21 days followed by Doxorubicin (20mg/kg intraperitoneally) on 21^st day.
• Group 4 and 5 These groups received aqueous extract of Hibiscus rosa sinensis orally in 2 graded doses (250 & 500mg/kg) for 21 days and Doxorubicin (20mg/kg intraperitoneally) on the 21^st day.

After giving Doxorubicin (20mg/kg intraperitoneally) on 21^st day, rats were observed for 48 hours and then sacrificed. The albino rats were sacrificed using Ketamine (75mg/kg) anesthesia, given intraperitoneally. Blood sample was collected from abdominal aorta (5ml) for performing blood test i.e. Creatine kinase-MB(CK-MB), Lactate dehydrogenase (LDH), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT). Also the heart was dissected out for the purpose of taking histological samples. The cardiac and histological markers were measured and compared with control group and the group treated with a standard cardioprotective drug (Carvedilol).

HISTOPATHOLOGICAL EXAMINATION

The heart was excised from the animal and washed with normal saline. Whole of the heart was placed in 10% neutral formalin for 12-24 hours. It was then dehydrated and cleared with ethanol and xylene, respectively, followed by embedding in paraffin wax from which blocks were prepared. Sections of 5µm thickness were prepared from the blocks using microtome. These were processed in alcohol-xylene series and were stained with Harris Haematoxylin and Eosin stain and then subjected to histopathological examination

STATISTICAL ANALYSIS

Mean ± SE was calculated for each group to observe the general trend of the group. The statistical analysis was carried out using one way analysis variation (ANOVA) followed by Post-Hoc Test. P- values were estimated by referring to appropriate tables⁹.

OBSERVATIONS AND RESULTS

 The present study revealed increased levels of serum markers of cardiotoxicity such as CK-MB, LDH, AST and ALT following Doxorubicin administration (20mg/kg i.p). These findings were consistent with study done by El-Agamy DS et al., (2016)¹⁰. The myocardial tissue of saline-treated rats showed clear integrity of the myocardial cell membrane and an absence of inflammatory cell infiltration. In contrast, Doxorubicin-injected rats exhibited separation of cardiac muscle fibers and infiltration of inflammatory cells. Rats treated with Hibiscus rosa sinensis (250mg/kg/day p.o + DOX 20mg/kg i.p single dose) for 21 days demonstrated gentle widening of the muscle fibres with focal loss of myofibres, inflammation and absence of myonecrosis. Rats treated with Hibiscus rosa sinensis (500mg/kg/day p.o + DOX 20mg/kg i.p single dose) for 21 days showed no inflammation, no edema and almost normal cardiac architecture which was similar to that seen in Carvedilol treated groups.

Table 1: Effect of Carvedilol, Aqueous extract of Hibiscus rosa sinensis in their respective doses on Doxorubicin (DOX) induced changes in CK-MB, LDH, ALT and AST levels (n = 6).

^αp< 0.01 as compared to DOX treated group.

^βp < 0.001 as compared to DOX treated group.

^γp < 0.001 as compared to normal saline treated group.

GRAPH

SLIDE 1: NORMAL SALINE  GROUP

SLIDE 2: DOX TREATED

SLIDE 3:  CARVEDILOL TREATED

SLIDE 4: HRS 250mg/kg treated

SLIDE5:  HRS 500mg/kg treated

DISCUSSION

Cardiovascular diseases (CVD) are non-communicable diseases encompassing a range of conditions that affect the heart and blood vessels. One important factor associated with CVD is cardiotoxicity, which refers to damage to the heart caused by toxic substances. It typically manifests as electrophysiological disturbances and myocardial (heart muscle) damage, which ultimately leads to heart failure¹¹. Among cardiotoxic agents, chemotherapeutic drugs are particularly notable. A major group within this category is the anthracyclines, which includes the widely used anticancer drug doxorubicin (DOX). Doxorubicin-induced cardiotoxicity is a progressive condition that begins with injury to myocardial cells, followed by a reduction in left ventricular ejection fraction (LVEF). If not detected and managed early, it can lead to symptomatic heart failure.

The underlying mechanisms of doxorubicin cardiotoxicity are complex. A key contributor is the generation of reactive oxygen species (ROS), along with disturbances in iron metabolism and calcium (Ca²⁺) signalling. Free radicals are highly reactive for instance, hydroxyl radicals can cause damage to membrane phospholipids, DNA, Molecules and proteins, in which the former would result in the formation of peroxyl radicals. Thus, when there are insufficient natural antioxidant components in the body, such as superoxide dismutase (SOD) enzyme and glutathione peroxidase (GSH-px), an oxidative chain reaction may occur, resulting in tissue damage. A critical molecular target is topoisomerase 2β (Top2β), an enzyme involved in uncoiling DNA during replication, transcription, and recombination. Doxorubicin inhibits Top2β, leading to mitochondrial dysfunction, activation of cell death pathways, and accumulation of ROS, all of which contribute to cardiac damage¹².

Carvedilol is taken as standard drug in present study. Carvedilol is a third generation β receptor antagonist, used to treat symptomatic congestive heart failure. Carvedilol has antioxidant, anti inflammatory and membrane stabilizing properties, thereby reduces arterial blood pressure by decreasing vascular resistance and tone. Carvedilol also inhibits ROS mediated loss of myocardial contractility, stress induced hypertrophy, apoptosis and activation of neutrophils¹³. Because of these properties carvedilol is considered to be a standard drug for comparison of cardioprotective activity as evidenced by study done by Varshney P et al.,(2016)⁸.

In the present study, serum Creatinine phosphokinase-MB (CK-MB), Lactate dehydrogenase (LDH), Aspartate aminotransferase (AST), and Alanine aminotransferase (ALT) were used as biochemical markers for assessing cardiac injury. The myocardium is rich in diagnostic marker enzymes for cardiotoxicity, and when damaged, it releases its intracellular contents into the extracellular fluid. Therefore, serum levels of these marker enzymes reflect alterations in membrane integrity and permeability. Cytosolic enzymes CK-MB, LDH, AST, and ALT, which serve as diagnostic markers, leak from damaged tissue into the bloodstream when the cell membrane becomes permeable or ruptures. AST and ALT are important for the regulation of metabolism. Necrosis and inflammation increase AST levels in the heart and liver. In Doxorubicin-treated rats, ALT and AST levels were significantly elevated compared to normal rats. These enzymes are valuable in diagnosing Doxorubicin-induced myocardial cardiotoxicity.

Hibiscus rosa-sinensis contains numerous compounds including quercetin, glycoside, riboflavin, niacin, carotene, anthocyanin, anthocyanidin, malvalic acid, gentisic acid, margaric acid and lauric acid. Hibiscus rosa sinensis possess strong antioxidant properties¹⁴. Potential antioxidant mechanism is preventing oxidative damage to the myocardial tissues.

Hibiscus rosa sinensis has anti-inflammatory, anti-ulcer, anti-diabetic, nootropic, hepatoprotective, anti-hypertensive and hypolipidemic and many more activities. This species have various flavonoids, glycosides, alkaloids and various phytochemicals that makes it more important candidate for the researchers. Guddeti V et al demonstrated anti inflammatory activity of Hibiscus rosa sinensis by reducing the paw edema in rats against standard drug diclofenac due to antioxidant property of its constituents¹⁵. Notably, CK-MB levels were significantly lower in rats pretreated with Hibiscus rosa sinensis. Hibiscus rosa sinensis provided cardioprotection as indicated by the limited rise in serum markers of cardiac damage. Furthermore, Hibiscus rosa sinensis at 500 mg/kg conferred more cardioprotection than at 250 mg/kg..

A study by Mohammed HS et al., (2020) demonstrated a significant increase in LDH levels in rats treated with Doxorubicin 48 hours post treatment¹⁶. Similarly increased level of LDH was also noticed in the present study. Pretreatment with Hibiscus rosa sinensis significantly reduced the elevated LDH levels, indicating a reduction in the severity of cardiotoxicity.

The serum marker results correlated with histopathological observations in the myocardial tissue of animals treated with Doxorubicin, test drugs and normal saline. Another study by Gauthaman KK et al., (2006) on the Cardioprotective effect of the Hibiscus rosa sinensis flowers in an oxidative stress model of myocardial ischemic reperfusion injury in rat also showed cardioprotective activity, similar to the findings of the present study¹⁷. The exact cardioprotective mechanism of aqueous extract of Hibiscus rosa sinensis has not been evaluated in the present study but it can be postulated to be due to presence of phytochemicals constituents with antioxidant potential. However, comprehensive and extensive research, conducted over a larger sample size is needed to explore the pharmacokinetic and pharmacodynamic profiles of these extracts, which will provide the foundation for future clinical trials.

CONCLUSION

Doxorubicin, a drug belonging to anthracycline group, is widely used as chemotherapeutic agent for treatment of cancers like breast carcinoma, hodgkin lymphomas and solid tumors. Excessive formation of free radicals and oxidative stress resulting in serious cardiotoxicity limits its clinical use. A continuous search to overcome this problem is going on. In the present study it was found that pretreatment with aqueous extract of Hibiscus rosa sinensis significantly reduced the doxorubicin induced damage to rat myocardium. No significant adverse effects were seen among rats after the administration of aqueous extract of Hibiscus rosa sinensis for 21 days. This study also provides scope for further evaluation of Hibiscus rosa-sinensis using their hydroalcoholic extracts at different dose levels, assessing additional biochemical parameters, and extending the duration of the tests. This cardioprotective potential of Hibiscus rosa sinensis might be attributed to the antioxidant property of chemical compounds present in them and can serve as a good source for the production of a cardioprotective herbal medicines. However, an extended study using larger number of animals with removal of confounding factors like pretreatment cardiac enzymes levels, sequential administration of doxorubicin and subsequent analysis is required so that substantial data can be generated for facilitating further evaluation of these agents through clinical trials. Further molecular level of investigation could be done using different animal models and other biochemical parameters to elucidate the possible mechanism of action of Hibiscus rosa sinensis.

Acknowledgements

Authors would like to thank Dr Monica Sharma (Professor and HOD, Department of Pharmacology, L.L.R.M. Medical College, Meerut) and Dr Preeti Singh (Professor, Department of Pathology, L.L.R.M. Medical College, Meerut) for their cooperation and sincere help in this study.

Funding: No funding sources

Conflict of interest: None declared

REFERENCES

1. Mounika S, Jayaraman R, Jayashree D, Pravalika KH, Balaji A, Sadiya M, et al. A comprehensive review of medicinal plants for cardioprotective potential. Int. J. Adv. Pharm. Biotech. 2021;7(1):24–9.
2. Albakri A. Drugs-related cardiomyopathy: A systematic review and pooled analysis of pathophysiology, diagnosis and clinical management. Internal medicine and care. 2019;3(1):1-19.
3. Prabhakaran D, Jeemon P, Roy A. Cardiovascular disease in India- Current epidemiology and future directions. AHAIASA. 2016;133(16):1605–20.
4. Shah SM, Akram M, Riaz M, Munir N, Rasool G. Cardioprotective potential of plant-derived molecules: a scientific and medicinal approach. Dose-response. 2019;17(2):1559325819852243.

1. Parveen A, Babbar R, Agarwal S, Kotwani A, Fahim M. Mechanistic clues in the cardioprotective effect of Terminalia arjuna bark extract in isoproterenol-induced chronic heart failure in rats. Cardiovascular Toxicology. 2011;11(1):48-57.

6. Agrawal KK. A Review on Pharmacological Validation of Genus Hibiscus with Main Emphasis on Hibiscus rosa-sinensis. IJPPR. 2020;18(3):451-62.
7. Goldberg KH, Yin AC, Mupparapu A, Retzbach EP, Goldberg GS, Yang CF. Components in aqueous Hibiscus rosa-sinensis flower extract inhibit in vitro melanoma cell growth. Journal of traditional and complementary medicine. 2017;7(1):45-9.
8. Varshney P, Vishwakarma P, Sharma M, Saini M, Bhatt S, Singh G. Cardioprotective effect of Solanum nigrum against doxorubicin induced cardiotoxicity- an experimental study. International Journal of Basic and Clinical Pharmacology. 2016;5(3):748-53.
9. Ghosh MN. Fundamentals of experimental pharmacology. 6th ed. Kolkata. Hilton and Company; 2015: 243.
10. El-Agamy DS, Abo-Haded HM, Elkablawy MA. Cardioprotective effects of sitagliptin against doxorubicin-induced cardiotoxicity in rats. Exp Biol Med. 2016;241(14):1577–87.
11. World Health Organization. Cardiovascular diseases (CVDs). World Health Organization. 2023 [cited 2025 May 28]. Available from: https://www.who.int/health-topics/cardiovascular diseases#tab=tab_1.
12. Zhang S, Liu X, Khalfe TB, Lu LS, Lyu YL, Liu LF, et al. Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat Med. 2012;18(11):1639–42.
13. Dandona P, Ghanim H, Brooks DP. Antioxidant activity of carvedilol in cardiovascular disease. J Hypertension. 2007;25(4):731–41.
14. Puckhaber LS, Stipanovic RD, Bost GA. Analysis of flavonoid aglycones in fresh and preserved Hibiscus flower. Trends in New Crops and New Uses. 2002;556-63.
15. Guddeti V, Babu DM, Nagamani B, Teja MR, Sravani MS, Spandana CH. Evaluation of anti inflammatory activity of hibiscus rosa sinensis linn. flower extract in rats. IJPCBS. 2015;5(3):532-6.
16. Mohammed HS, Khadrawy YA, Monem AS, Amer HM, Sherbini TME, Rahman MAE. Formulated curcumin nanoparticles mitigate brain oxidative stress induced by reserpine in rats. J. Bio nano science. 2020;1866(5):290-6.
17. Gauthaman KK, Saleem STM, Thanislas TP, Prabhu VV, Krishnamoorthy KK, Devaraj SN, Somasundaram SJ. Cardioprotective effect of the Hibiscus rosa sinensis flowers in an oxidative stress model of myocardial ischemic reperfusion injury in rat. BMC Complementary and Alternative Medicine. 2006;6(1):32.