We established in vivo models of isoproterenol-induced myocardial infarction (MI) to determine the cardioprotective effect of Spirulina platensis (Spi), Momordica charantia fruit (MC) and their combination. MI was induced in six groups of Male Wistar albino with isoproterenol, and cardioprotection was evaluated by measuring SGOT, SGPT, LDH, CK, CK-MB with commercially available test kits and analyzing histopathology. Online PASS was used to predict the mechanism of action of a marker compound in Spi and MC. Rat serum levels of LDH, SGOT, CK-MB, and CK were significantly reduced by the Spi and MC combination extract (P < 0.05). The combination extract at a dose of 50:50 mg/kg body weight preserve the integrity of the myocardial cell membrane. Identified compounds by LC-MS/MS in the ethanol extract of Momordica charantia are (3β,16α-Dihydroxy-lanosta-8,24-dien-21-oic acid), Eclalbasaponin II, Epianhydrobelachinal, Epianhydrobelachinal, Paeonenolide F, Ziyu glycoside II. Identified compounds by LC-MS/MS in Spirulina platensis extract are d-Lirioferine, Lycopodine, Yuanhunine, Candidate Mass C18H26N2O5 and Candidate Mass C36H38N4O5. In PASS analysis, Phycocyanobilin’s cardioprotective mechanism of action is predicted to be kinase inhibitor, cytoprotectant, and platelet aggregation inhibitor, with Probable activities (Pa) of 0.06, 0.53, and 0.443, respectively. The Pa values for cholesterol antagonist, proliferative disease treatment, nitric oxide scavenger, and anti-inflammatory agents in Momordicoside A are respectively 0.841, 0.666, 0.59, and 0.55. These findings suggest that the cardioprotective activity of the combination of Spi and MC extracts at a dose of 50 mg/kg was synergistic.

cardioprotective, isoproterenol, LC-MS/MS, Momordica charantia, Spirulina platensis

Myocardial infarction (MI), also known as a heart attack, is a disease caused by prolonged ischemia that causes irreversible death or necrosis of cardiac muscle (Mythili and Malathi 2015). According to WHO data, heart attacks and strokes account for up to 85 percent of all cardiovascular disease-related fatalities. According to estimates, 17.9 million deaths worldwide from cardiovascular disease occurred in 2016, contributing for 31% of all fatalities (WHO 2022). In 2030, cardiovascular disease is projected to continue to be the leading cause of death, affecting 23.6 millions of people (Chadwick Jayaraj et al. 2019).

A heart attack causes more severe damage to the heart, which can result in a decline in cardiac function and death (CDC 2021). Currently, there is no drug that can prevent heart organ damage; therefore, the goal of this research is to discover alternative drugs that can prevent more severe heart damage (cardioprotective) so that heart function can be preserved and death from heart attack can be prevented.

As a beta – adrenergic agonist, isoproterenol (ISO) can induce infarction by inducing oxidative stress in the myocardium and necrosis similar to heart muscle infarction Additionally, it has been demonstrated that ISO generates free radicals and promotes lipid peroxidation, both of which are thought to contribute to the long-term deterioration of cardiac membranes. To prevent or even treat myocardial ischemia, novel medications are required because current treatments have a minimal impact on survival and recurring costs (Murugesan et al. 2011).

Spirulina platensis (Spi) are cyanobacteria containing a variety of compounds including proteins, lipids, polysaccharides, and pigments. Phycocyanobilin (PC) is the primary pigment responsible for Spirulina’s green-blue color (Fernández-Rojas et al. 2014). An imbalance between the antioxidant system and free radical material will result in inflammation and a total number of diseases, including atherosclerosis, cancer, neurodegenerative diseases, and diabetes mellitus. Internal and external sources of free radicals can have a significant impact on the biological molecules of the human body. In addition to Phycocyanobilin, Spirulina platensis contains compounds that contribute to its antioxidant capacity, including chlorophyll, carotenoids, phenolic compounds, polysaccharides, fatty acids and vitamins (Asghari et al. 2016). In human skin fibroblast cell lines, Spirulina can enhance migration and proliferating (Liu et al. 2019). Spirulina extract also promotes re-epithelialization and enhance the vascularity of wound healing (Bahei-Eldin et al. 2017).

The Cucurbitaceae family, of which Momordica charantia (MC) is a member, has a wide geographical distribution, and this plant contains numerous phytochemicals including triterpenes, flavonoids, and saponins (Bhowmik 2010). Several reports of M. charantia’s biological activity, including anti-inflammatory, anti-carcinogenic, hepatoprotective effect, anti-viral, anti-tumor, immunomodulating, anti-mutagenic, anti-ulcer, anti-lipolytic, and antifertility properties (Jia et al. 2017). Traditionally, MC has been used to treat conditions such as hyperglycemia, cancer disease, a variety of infection disorders (Bhowmik 2010). In this study, we evaluated the prospective cardioprotective effects of Spirulina platensis extract, bitter melon (Momordica charantia) fruit extract, and their combination.

Myocardial infarction is strongly associated with mortality and morbidity (Chan and Ng 2010). MI refers to the loss of myocardial cells caused by prolonged ischemia. Reduced cellular glycogen and loosened myofibrils can occur within 10 to 15 minutes of the onset of ischemia (Thygesen et al. 2018). Cardioprotection incorporates all techniques and strategies that aid in maintaining heart healthy by reducing or preventing myocardial damage (Kubler and Haass 1996). Rise/fall of cardiac troponin detection can be used to asses MI condition and one of the following: 1) ischemia–related symptoms, 2) new pathological Q waves, 3) electrocardiogram alterations of new ischemia (Chan and Ng 2010).

Both the production of free radicals and the induction of lipid peroxidation by isoproterenol are likely to contribute to the long-term deterioration of cardiac membranes (Murugesan et al. 2011). After isoproterenol-induced myocardial infarction, elevated troponin I and urea, leukocyte and neutrophil, SGOT concentration, decreased catalase enzyme activity, and increased glutathione levels were all observed in the myocardium (Lobo Filho et al. 2011).

In addition to symptoms of ischemia, abnormal cardiac biomarkers serve as supporting evidence for diagnosing MI (Thygesen et al. 2018). In our study, the assessment of cardioprotective characterization was seen from changes in biomarker levels in MI conditions. Biomarker levels including SGPT, SGOT, CK, CK-MB and LDH were anticipated to decrease after 14 days of pretreatment with extracts. We found that the administration of ISO has no effect on the level of SGPT. 100 mg/kg body weight and 300 mg/kg body weight of MC fruit extract only reduces LDH levels. In the past, the AST (SGOT) and LDH enzyme were originally employed to diagnose MI. However, because these enzymes lacked the optimal cardiac indicators, they were never utilized.

However, because they are limited and simple to assess in hospitals, along with the patient’s medical history, they can provide doctors with a general understanding. CK-MB and CK enzyme measurements are indeed useful in the diagnosis of AMI (Aydin et al. 2019)increase the quality of life of patients, and decrease health expenditure in many countries. In this study, the advantages and disadvantages of the enzymatic and nonenzymatic biomarkers used in the diagnosis of patients with AMI are given in historical sequence, and some candidate biomarkers – hFABP, GPBB, S100, PAPP-A, RP, TNF, IL6, IL18, CD40 ligand, MPO, MMP9, cell-adhesion molecules, oxidized LDL, glutathione, homocysteine, fibrinogen, and D-dimer procalcitonin – with a possible role in the diagnosis of AMI are discussed.\n\nMethods\nThe present study was carried out using meta-analyses, reviews of clinical trials, evidence-based medicine, and guidelines indexed in PubMed and Web of Science.\n\nResults\nThese numerous AMI biomarkers guide clinical applications (diagnostic methods, risk stratification, and treatment. Statistical tests revealed no significant difference between the two concentrations of MC fruit extracts in their ability to reduce LDH levels, indicating that increasing the dose had no effect.

The organs with the highest concentration of LDH include the kidney, skeletal muscle, liver, and heart. Within six to twelve hours of the onset of chest pain, it rises, peaks within one to three days, and returns to normal within eight to fourteen days. Infarct size is correlated with total CK and CK-MB levels, which are important prognostic markers. This reaction is catalyzed by the enzyme CK, which contributes to the conversion of creatine and ATP into creatine phosphate and ADP. Approximately 20% of myocardium contains CK in the MB form (CK-MB), making the measurement of CKMB very useful for supporting data for the diagnosis of AMI (Aydin et al. 2019)increase the quality of life of patients, and decrease health expenditure in many countries. In this study, the advantages and disadvantages of the enzymatic and nonenzymatic biomarkers used in the diagnosis of patients with AMI are given in historical sequence, and some candidate biomarkers – hFABP, GPBB, S100, PAPP-A, RP, TNF, IL6, IL18, CD40 ligand, MPO, MMP9, cell-adhesion molecules, oxidized LDL, glutathione, homocysteine, fibrinogen, and D-dimer procalcitonin – with a possible role in the diagnosis of AMI are discussed.\n\nMethods\nThe present study was carried out using meta-analyses, reviews of clinical trials, evidence-based medicine, and guidelines indexed in PubMed and Web of Science.\n\nResults\nThese numerous AMI biomarkers guide clinical applications (diagnostic methods, risk stratification, and treatment.

Single extract of Spirulina platensis reduces CK and LDH levels, whereas single extract of MC fruit reduces only LDH levels. The evaluation of MC fruit extract at a dose of 300 mg/kg b. wt. revealed no significant difference compared to MC fruit extract at 100 mg/bw (data not shown). According to our findings regarding the cardioprotective effects of Spirulina platensis, MC fruits, and their combination extracts, the combination extracts of Spirulina platensis and MC fruits provided the greatest cardioprotective effect in rats with isoproterenol-induced MI. SGOT, CK, CK-MB, and LDH levels can be reduced by the combination of Spirulina platensis and MC fruit extracts. Natural product combinations are frequently advocated for medical objectives on the grounds that their “synergistic” interactions render them more potent than pure substances (Caesar and Cech 2019) approximately 18% of the U.S. population uses natural products (including plant-based or botanical preparations. Cardioprotective agents function as a pretreatment to reduce the incidence of necrosis or safeguard the heart in the event of a myocardial infarction (MI) (Chan and Ng 2010). Two extracts of Spirulina platensis and MC fruits may have a synergistic effect, particularly as cardioprotective, when combined.

The term “cardioprotective medications” refers to substances that protect the heart from the harmful effects of acute myocardial ischemia. Therefore, they must first prevent sudden cardiac mortality due to local myocardial ischemia; then, if possible, they must minimize the size of the developing infarct; and finally, they must reduce the likelihood of persistent infarct or infarct extension (Szekeres, 1987)such as sudden coronary death (SCD. Histological examination revealed that the myocardium cells in the negative group had excellent integrity, whereas in the positive control group administered isoproterenol, the myocardium cells exhibited leukocyte infiltration. In terms of cell integrity, integrity of the cell nucleus, and minimal leukocyte infiltration, the dose of 100 mg/kg b.w. of MC in the test group and the combination group produced the greatest results.

Using in silico PASS analysis prediction, we then attempted to determine the mechanism underlying the cardioprotective effect of Spirulina platensis and MC fruits extract. Prediction of the mechanism of action based on the main constituents of Spirulina platensis (Phycocyanobilin) and MC fruits extract (Momordicoside A). Potential cardioprotective activities in Phycocyanobilin include kinase inhibitor, cytoprotectant, and platelet aggregation inhibitor. Among Momordicoside A’s putative cardioprotective activities are cholesterol antagonist, treatment for proliferative diseases, nitric oxide scavenger, and anti-inflammatory.

Myocardial infarction is associated with an inflammatory response, which is required for healing and scar formation (Frangogiannis et al. 2002). Myoglobin (Mb) is a key intracellular nitric oxide (NO) scavenger that regulates the antioxidant effect in coronary flow and cardiac contractility, according recently published experimental evidence. Low Mb level and increased iNOS activity have been observed in patients with heart failure. Therefore, pathophysiology of prevalent disease states may be influenced by the loss of Mb and resulting decrease in O₂ supply and NO scavenging capacity in failing hearts (Merx et al. 2005). Commonly, the formation of fibrous scar tissue in the infarct zone results in chronic problems and functional deficiencies. Fibroblast stimulation and proliferation are essential for maintaining heart integrity and function shortly after cardiac injury (Shen et al. 2015). In addition to its heart-protective anti-atherogenic properties, a growing body of evidence from clinical and experimental investigations indicated that HDL (also known as a cholesterol antagonist) has a variety of positive health impacts. HDL can protect the myocardium by acting as an energy source, thereby supporting anti-inflammatory and antioxidant mechanisms, as well as NO-mediated vasodilators (Nagao et al. 2018).

The primary component of Spirulina platensis, Phycocyanobilin, has potential cardioprotective properties such as kinase inhibitor, cytoprotectant, and platelet aggregation inhibitor. Study from (Patel et al. 2016) show that Ibuprofen has a cardioprotective effect on rats with isoproterenol-induced myocardial infarction by inhibiting RhoA/Rho kinase, indicating that kinase inhibitor can be considered in the development of cardioprotective agents. As reported, cytoprotective drugs such as urocortin (Davidson et al. 2009), 7-oxoPGI2 (Szekeres 1987) such as sudden coronary death (SCD, and trimetazidine have cardioprotective effects (Tallarico et al. 2003). Most cytoprotective drugs function by stabilizing cell membranes. They protect cellular integrity from the harmful effects of toxic substances or metabolites by preventing or reducing harmful effects such as electrolyte changes, cell swelling, lysosomal enzyme release, and free radical release. The mechanism for cell membrane stabilization contributes to cytoprotection (Szekeres 1987).

During the early hours following myocardial ischemia, damaged cardiac cells can release adenosine, opioids, and bradykinin, which enhance myocardial survival by activating the G protein signaling pathway (Liu et al. 2011). According to prediction in PASS analysis, Phycocyanobilin has the potential to inhibit platelet aggregation which can be advantageous. Only active thrombolytic treatment, angioplasty, or surgical revascularization can restore blood flow in the case of developed thrombi. If there is sufficient time and the injury is not life-threatening, ischemia-induced spontaneous development and expansion of coronary anastomoses may be able to reduce the infarct size (Szekeres 1987).

Based on the results of the LC-MS/MS analysis, five compounds have been identified, but neither in vitro nor in vivo testing has been conducted. Therefore, it cannot be determined whether these compounds are cardioprotective. Based on marker compounds found in Spirulina platensis and Momordica charantia extracts, we conducted a PASS analysis test to make a prediction.

In conclusion, we were able to demonstrate that combination of Spirulina platensis and Momordica charantia fruits extract has cardioprotective effect with attenuated cardiac injury biomarker in rats with isoproterenol-induced myocardial infarction. Histological evaluation reveals that the 100 mg/kg bw MC test group and the combination group maintain cell integrity, nucleus integrity, and minimal leukocyte infiltration. Phycocyanobilin in Spirulina platensis and Momordicoside A in Momordica charantia fruits possess a cardioprotective mechanism as a kinase inhibitor, cytoprotectant, platelet aggregation inhibitor, cholesterol antagonist, treatment for proliferative diseases, nitric oxide scavenger, and anti-inflammatory. The administration of a solitary extract did not have a satisfactory cardioprotective effect, but administration as a combination yielded the best results. However, the dose increase had no effect on the activity of the substance.

Ethical Concerns the Animal Research Ethics Committee of the School of Pharmacy at Institut Teknologi Bandung granted ethical approval for the investigation (No. 05/KEPHP-ITB/03-2021).

The authors declare publication of this article does not involve any conflicts of interest. Authors confirm that the paper was original.

We are grateful to the Research Program at Institut Teknologi Bandung for their financial support of this project (SF.PN-6-06-2022).