INTRODUCTION:

Diabetes and heart complications have become significant public health issues. The primary causes of death and disability in diabetic patients include myocardial infarction (MI), angina, ischemic heart failure, and arrhythmias¹^,². Acute myocardial necrosis occurs due to an imbalance between the heart muscle's oxygen demand and coronary blood flow. Factors such as increased free radical production, apoptosis, inflammation, and persistent DNA damage contribute to the development of MI³^,⁴.

The relationship between diabetes and cardiovascular complications is largely attributed to oxidative stress and heightened apoptosis. Chronic hyperglycemia and advanced glycation end products trigger the formation of superoxide anions and reactive oxygen species (ROS), raising the risk of cardiovascular disease⁵^,⁶. Oxidative stress can cause damage to biomolecules and activate harmful signaling pathways, leading to cell dysfunction and death⁷.

The treatment and management of diabetic patients typically involve the use of beta blockers, angiotensin-converting enzyme inhibitors (ACE inhibitors)/angiotensin II receptor blockers (ARBs), nitrates, and antithrombotic medications⁸. These drugs help relieve myocardial infarction (MI) symptoms by improving blood flow to the heart and protecting it from apoptotic damage. However, despite these interventions, the mortality rate for acute diabetes remains over 30%. Additionally, the effectiveness of current medications is limited and presents significant drawbacks⁹. As a result, there is a need for a new drug or agent with minimal side effects to prevent and treat heart disease while also helping regulate blood glucose levels¹⁰^,11.

The herbonanoceutical formulation of tamoenju (Hibiscus surattensis L.) leaf extract is developed as a self-nano emulsifying system, representing an advancement in creating a drug with cardioprotective properties. This formulation is intended to serve as an adjuvant therapy for managing diabetes-related complications. Previous research has proven the herbonanoceutical activity of tamoenju leaf extract as a very strong antioxidant and has activity as an angiotensin converting enzyme inhibitor¹².

However, no scientific investigation has yet been conducted on the effect of herbonanoceutical tamoenju leaf extract on diabetes-induced cardiac dysfunction, which is also driven by oxidative stress. Therefore, this study was carried out to examine the anti-hyperglycemic activity of the herbonanoceutical of tamoenju leaf extract and its impact on diabetes-induced cardiac dysfunction and biochemical injury markers.

MATERIALS AND METHODS:

Materials:

Virgin coconut oil (VCO), tween 80, and propylene glycol (Bratachem, Indonesia). Streptozotocin, nicotinamide and isoproterenol were purchased from Sigma-Aldrich. The kits for the determination of creatine kinase (CK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), SGOT and SGPT purchased from Biosystems Diagnostic, SOD and MDA reagen kit from Bioassay Technology Laboratory.

Sample:

Herbonanoceutical from tamoenju leaf extract (a formula optimized in previous research) underwent characterization tests, including particle size, zeta potential, polydispersity index, pH, and viscosity analysis. The herbonanoceutical from tamoenju leaf extract was analyzed using Fourier Transform Infrared Spectroscopy (FT-IR) to identify compounds based on frequencies and wavenumbers that indicate functional groups and to determine possible interactions between active pharmaceutical ingredients and excipients. Scanning was performed over a wavenumber range of 4000-600 cm⁻¹ using FT-IR.

Animals:

Adult male Sprague Dawley rats were used, ages 2-3 months and weighing 150-250 grams. Exclusion criteria include white mice that are physically disabled, appear sick, or die during the study. They were kept under natural light and a dark cycle, at constant room temperature (25± 5°C) and humidity (55±5%). The animals were fed with standard AD2 feed and water ad libitum. The experimental protocols were approved by the Medical and Health Research Ethics Committee (No. 864/UN28.1.30/KL/2024) of the Faculty of Medicine, Tadulako University, Palu, Central Sulawesi.

Induction of diabetes and myocardial infarction:

Rats were acclimated for 7 days before testing began. Diabetes was induced in rats by a single intraperitoneal injection of streptozotocin (50 mg/kg) administered 15 minutes after nicotinamide administration (120 mg/kg). STZ was dissolved in 0.1 M citric buffer (pH 4.5), and nicotinamide (120 mg/kg BW) was dissolved in aqua pro injection. On the seventh day after STZ and nicotinamide injection, rats with blood glucose levels above 250 mg/dL were selected as diabetic rats for further study. We randomly divided the diabetic rats into different groups, with six rats in each group. Isoproterenol (85 mg/kg BW) was administered via intraperitoneal route to diabetic rats for two consecutive days (days 13 and 14) for the induction of experimental myocardial infarction¹³^,¹⁴. Table 1 outlines the groups and their respective treatments.

Table 1. Grouping of animals

Groups	Treatments given
Group 1 Normal control (NC)	Vehicle (basis herbonanoceutical) for 14 days
Group 2 Diabetic isoproterenol-treated (STZ+ISO)	Vehicle (basis herbonanoceutical) for 14 days and isoproterenol (85 mg/kg BW) was also administered intraperitoneal to the rats on days 13 and 14.
Group 3 Diabetic isoproterenol-treated rats administered with metformin and atenolol (STZ+ISO+Met+At)	Rats received 45 mg/kg/BW/day of metformin and 9 mg/kg BW/day atenolol orally for 14 days. On days 13 and 14, rats were injected intraperitoneal with isoproterenol (85 mg/kg BW).
Group 4 Diabetic isoproterenol-treated rats administered with 25 mg/kg BW herbonanoceutical of tamoenju leaves extract (STZ+ISO+SNASET 25)	Rats received 25 mg/kg/BW/day herbonanoceutical of tamoenju leaves extract orally for 14 days. On days 13 and 14, rats were injected intraperitoneal with isoproterenol (85 mg/kg BW).
Group 5 Diabetic isoproterenol-treated rats administered with 50 mg/kg herbonanoceutical of tamoenju leaves extract (STZ+ISO+SNASET 50)	Rats received 50 mg/kg/BW/day herbonanoceutical of tamoenju leaves extract orally for 14 days. On days 13 and 14, rats were injected intraperitoneal with isoproterenol (85 mg/kg BW).

Blood sample collection:

Blood glucose level was measured using glucometer and glucose standard strip/kits (Easy Touch). Test samples in rat blood were collected via the intracardiac route. After 24 hours from the last administration of isoproterenol, blood samples were drawn from the heart after the rats were anesthetized with ketamine. The blood was centrifuged at 12,000 rpm for 10 minutes to obtain serum. The obtained serum was stored at -20°C until needed to determine the AST, ALT, CK, CK-MB, and LDH levels in U/L units. The levels of AST, ALT, CK, CK-MB, and LDH obtained were interpreted to evaluate cardioprotective activity.

Measurement of SOD and MDA levels in rat cardiac serum:

Measurement of SOD and MDA levels was performed using the Bioassay Technology Laboratory (BT Lab) rat ELISA kit, catalog number E0168Ra for SOD and E0156Ra for MDA, following the kit’s guidelines. A 50 μL aliquot of the standard solution or sample was added to each well, followed by 50 μL of Biotinylated Detection Ab. The mixture was incubated at 37°C for 45 minutes and then aspirated. A 350 μL wash buffer was added, and the wells were washed three times. Next, 100 μL of HRP Conjugate Working Solution was added, incubated at 37°C for 30 minutes, aspirated, and washed five times. Subsequently, 90 μL of substrate reagent was added and incubated at 37°C for 15 minutes. Finally, 50 μL of stop solution was added. The absorbance of each sample was measured using a microplate reader at λ = 450 nm. Linear regression was performed using the standard absorbance values, and the sample absorbance was then measured against the standard curve, with units of ng/mL for SOD and nmol/mL for MDA.

Histopathological Analysis:

Following the conclusion of the study, histopathological analysis was performed. Serial heart tissue sections were prepared using a microtome, stained with hematoxylin and eosin, and examined under a microscope. The histological structure of the heart is examined under a Pro Histo Biological Microscope Pro 31W at 400x magnification. Infarction events are scored as follows: a score of 0 if there is no damage, 1 if less than 25% of the tissue is damaged, 2 for 25-50% damage, 3 for 50-75% damage, and 4 for 75- 100% damage. Digital images of the stained tissues were captured using Mv Image software. The pathologist conducting the examination and microscopy analysis was unaware of the treatment group assignments

Statistical Analysis:

The data are expressed as mean ± standard error of the mean (SEM). Statistical significance was assessed using GraphPad Prism software. A one-way analysis of variance (ANOVA) was performed, followed by a Duncan post hoc test. A p-value of less than 0.05 was regarded as statistically significant.

RESULT AND DISCUSSION:

Characterization of herbonanoceutical extract of tamoenju leaves:

This study aims to prepare, characterize, and integrate tamoenju leaf extract (Hibiscus surattensis L.) into a nanoemulsion system with potential as cardioprotective in animal models of myocardial infarction and diabetes. The results of the characterization of the Tamoenju leaf extract nanoemulsion or herbonanoceutical made with a composition of 0.1% tamoenju leaf extract, 10% VCO oil phase, a combination of Tween 80 surfactants and propylene glycol 2:1 as much as 90% are presented in Table 2.

Table 2. Characterization of herbonanoceutical extract of tamoenju leaves

Characteristic test	Herbonanoceutical extract of tamoenju leaves
pH	7.8 ± 0.1
Viscosity (cP)	131.2 ± 2.1
Particle Size (nm)	2.98 ± 0.32
Zeta Potential (mV)	-26.88 ± 1.29
Polydispersity Index	0.37 ± 0.03

The pH test was conducted to ensure the compatibility of the herbonanoceutical formulation's pH with the gastrointestinal pH, enabling absorption by the stomach. The pH value plays a crucial role in determining the effectiveness and stability of the active substances in the formulation. The pH value of the herbonanoceutical from tamoenju leaf extract is approximately 7.8 ± 0.1. This aligns with the ideal pH range for nanoemulsion formulations intended for oral administration, which is between pH 6.0-8.0¹⁴^,¹⁵.

Viscosity testing was performed using a Brookfield viscometer to measure the thickness of the nanoemulsion formulation. The viscosity of nanoemulsions produced through high-energy emulsification methods typically ranges from 10 to 2000 cP¹⁶. The test results indicated that the herbonanoceutical from tamoenju leaf extract meets the specified viscosity requirements.

Particle size is an essential parameter in the production of nanoemulsions, as one of their defining characteristics is a particle size range of 1–100 nm¹⁷. Particle size measurement was conducted using a PSA instrument to determine the particle size of the nanoemulsion formulation. This demonstrates that the formulation meets the standards for good nanoemulsion production, with particle sizes <100 nm.

The determination of zeta potential value aims to assess colloidal stability or the physical stability of the nanoemulsion. The zeta potential indicates the surface charge of the nanoemulsion globules; similar charges create repulsive forces between globules, preventing flocculation (the aggregation of small globules into larger ones)¹⁸. The desired zeta potential range is between -25 mV to -30 mV, as this range enables the formation of an energy barrier between droplets, resulting in a more stable nanoemulsion¹⁹. The zeta potential of the herbonanoceutical from tamoenju leaf extract exceeded -25 mV, indicating that the formulation is considered stable.

The polydispersity index (PDI) indicates the uniformity of droplet sizes in the formulation. A PDI value of <0.5 suggests that the smaller the PDI, the more uniform the droplet sizes in the formulation²⁰. The results indicate that the herbonanoceutical from tamoenju leaf extract has smaller and more homogeneous particle sizes.

FTIR (Fourier Transform Infrared Spectroscopy) characterization of nanoemulsions aims to identify chemical interactions between components in the formulation, such as the active ingredient, surfactants, and oil²⁰^,²¹. FTIR can be used to ensure that there are no significant changes to the chemical structure of the active ingredient after the formulation process, as well as to verify the stability and compatibility of ingredients in the nanoemulsion²². Additionally, FTIR helps confirm the presence of specific functional groups that contribute to pharmacological activity and formulation stability. The FTIR spectrum of tamoenju leaf extract, base, and herbonanoceutical from tamoenju leaf extract can be seen in Figure 1.

The FTIR spectrum of the nanoemulsion formed from tamoenju leaf extract and the herbonanoceutical base shows the following peaks: (1) Peak around 3400 cm⁻¹: This peak indicates the presence of -OH groups from both the extract and the nanoemulsion base, suggesting that hydrogen bonding interactions between the components in the nanoemulsion do not significantly alter the structure of the hydroxyl group. (2) Peak around 2900 cm⁻¹: This peak still indicates the presence of aliphatic C-H groups from the oil or surfactant in the nanoemulsion formulation, meaning the hydrocarbon chain remains intact in the formulation. (3) Peak around 1700 cm⁻¹: The visible carbonyl (C=O) group indicates that active components from the extract, such as phenolic compounds or organic acids, remain present and stable in the nanoemulsion. (4) Peak around 1600 cm⁻¹: This indicates the presence of aromatic C=C double bonds, likely derived from phenolic compounds in the extract, suggesting that the pharmacological activity of the active compounds may still be preserved. (5) Peak around 1050–1150 cm⁻¹: The ether (C-O-C) groups from the surfactant or glycosides in the extract are still visible, indicating compatibility and stability between the leaf extract and the base ingredients in the nanoemulsion.

In the FTIR spectrum, specific peaks indicate the presence of functional groups in the tested compounds. Using the FTIR method, it can be confirmed that there is no degradation of the active ingredient during nanoemulsion formulation. The spectra of tamoenju leaf extract and the nanoemulsion show significant similarities, indicating that the active ingredient remains stable, contributing to improved solubility and bioavailability. The tamoenju leaf extract contains hydroxyl (-OH), carbonyl (C=O), and aromatic C=C double bonds, indicating the presence of active compounds such as phenolics, flavonoids, or tannins, which are important for pharmacological activity. The herbonanoceutical base contains components such as hydrocarbon chains, hydroxyl groups, and carbonyl groups from oils and surfactants that form the emulsion. The formed nanoemulsion retains the functional group characteristics of the tamoenju leaf extract without significant changes, indicating that the active ingredients remain stable and compatible within the nanoemulsion formulation.

(a)

(b)

(c)

Figure 1. FTIR spectrum (a) tamoenju leaves extract (b) herbonanoceutical base (c) herbonanoceutical from tamoenju leaves extract

Effects of pharmacological interventions on blood glucose levels in diabetic and myocardial infarction:

The fasting blood glucose levels of the white RATS were measured four times. The first measurement was taken on day 0, just before the induction of streptozotocin and nicotinamide, to ensure that the RATS to be induced did not have diabetes mellitus (DM). On day 7, the second measurement was conducted to confirm the success of DM induction in the RATS. The third and fourth measurements were taken on days 14 and 21 to assess the effects of treatment on each group. The average fasting blood glucose levels for each group are shown in Figure 2.

Figure 2. Effects of pharmacological interventions on blood glucose levels in different groups

The data are expressed as mean± SEM (n = 6). Different alphabets on the same day indicate significant differences between treatment groups (p<0.05)

The results of blood glucose level measurements in Fig.2 show that the average fasting blood glucose levels before STZ and NA induction on day 0 of each treatment group were not much different and within normal limits (<126 mg/dL). Examination of fasting blood glucose levels on the 7th day after induction showed an increase in blood glucose levels (>250 mg/dL) in the STZ-NA induced group so that it can be concluded that STZ-NA injection was successful as an induction of diabetes mellitus model rats in all test animals in this study.

The fasting blood glucose levels measured on days 14 and 21 showed that the average fasting blood glucose levels in the normal control group did not increase compared to the measurements on days 0 and 7. In the diabetic+isoproterenol group, which was only given the herbonanoceutical base, there was no decrease in the average fasting blood glucose levels. The groups treated with the comparator metformin+atenolol and herbonanoceutical tamoenju leaf extract at doses of 25 and 50 mg/kg body weight showed a decrease in the average fasting blood glucose levels.

The diabetic+isoproterenol+metformin+atenolol group showed a noticeable reduction in fasting blood glucose levels from day 14 onward in this group. The combination of metformin and atenolol treatment helped manage hyperglycemia, as evidenced by the decreasing trend of glucose levels compared to the diabetic control group without treatment. Rats treated with a dose of 25 mg/kg BW herbonanoceutical of the tamoenju leaf extract showed moderate reductions in blood glucose levels by day 14, with further reductions by day 21. Although the glucose levels did not drop to normal, the treatment demonstrated a glucose-lowering effect compared to the untreated diabetic group. The higher dose of herbonanoceutical tamoenju leaf extract resulted in a more pronounced decrease in blood glucose levels, particularly by day 21. The glucose levels in this group were lower than those in the 25 mg/kg group, suggesting a dose-dependent effect of the herbonanoceutical extract in managing hyperglycemia.

Effects of pharmacological interventions on cardiac and liver injury markers diabetes and myocardial infarction rats

(a)

(b)

(c)

(d)

Figure 3. Effect of pharmacological interventions on the cardiac and liver injury markers in myocardial infarcted diabetic rats. (a) CK; (b) CK-MB; (c) LDH; (d) SGOT and SGPT. The data were expressed as mean ± SEM and analyzed using one-way analysis of variance (ANOVA) followed by Tukey post hoc test (p<0.05)

The STZ+ISO group in Fig.3 shows significantly higher CK, CK-MB, and LDH levels, indicating a possible association with myocardial (heart muscle) and tissue damage. The treatment groups (STZ+ISO+SNASET 25 and STZ+ISO+SNASET 50) effectively reduce elevated CK, CK-MB, and LDH levels caused by STZ+ISO-induced damage. This suggests a protective effect of the SNASET treatment, with a dose-dependent response where the higher dose (50) seems to provide better protection against muscle and tissue damage. Statistical analysis reveals significant differences between the groups, highlighting the efficacy of the treatment.

Streptozotocin (STZ) is used in research to induce diabetes in animals because it destroys pancreatic beta cells, which are responsible for producing insulin. When these beta cells are destroyed, the animals develop hyperglycemia²³^,²⁴. Isoproterenol (ISO) is used in research to induce experimental heart damage or myocarditis, as it is a beta-adrenergic agonist that increases sympathetic activity, leading to an elevated heart rate and stronger heart contractions. Excessive ISO use can cause oxidative stress and damage to the myocardium (heart muscle). The combined use of metformin and atenolol in this study is based on their mechanisms of action, which can reduce liver damage associated with oxidative stress and inflammation due to diabetes or cardiovascular stress. Metformin is used to counteract the effects of hyperglycemia induced by STZ, thereby alleviating stress on the body's organs, including the liver. Additionally, metformin has antioxidant and anti-inflammatory properties that may help protect the liver from damage caused by oxidative stress or inflammation under hyperglycemic conditions.

Both SGOT and SGPT are markers for liver damage. Higher levels typically indicate more severe liver stress or damage²⁵. The STZ+ISO group shows elevated levels of SGOT and SGPT, suggesting significant liver damage due to the induction of STZ and ISO. The normal control group has much lower enzyme levels, which is expected since it represents the normal condition without any induced stress. In the STZ+ISO+Met+At group, the SGOT and SGPT levels are lower than in the STZ+ISO group, indicating that the combination of Metformin and Atenolol protects liver function.

The STZ+ISO+SNASET 25 and STZ+ISO+SNASET 50 groups also show reduced SGOT and SGPT levels compared to the STZ+ISO group, with the higher dose (50 mg/kg) of SNASET appearing to be more effective in reducing liver enzyme levels. The STZ+ISO+SNASET 50 group shows a more pronounced reduction in enzyme levels than the STZ+ISO+SNASET 25 group, indicating a dose-dependent protective effect. These findings could point to potential therapeutic approaches for mitigating liver damage in conditions involving STZ and ISO-induced stress.

Effect of herbonanoceutical from tamoenju leaves extract on antioxidant parameters:

Streptozotocin and isoproterenol induction significantly decreased SOD levels and increased MDA levels, indicating potential oxidative damage to the cell membrane²⁶. Herbonanoceutical therapy from tamoenju leaf extract significantly increased endogenous antioxidant levels and decreased lipid peroxidation compared to the diabetic isoproterenol group. Based on the data in Fig.4, these effects are attributed to the active compounds in the herbonanoceutical extract of tamoenju leaves, which act as antioxidants and antidiabetics agents.

(a) (b)

Figure 4. The effect of herbonanoceutical from tamoenju leaves extract on oxidative stress in different groups (a) SOD (b) MDA

Histopathological analysis:

The histopathological results differed signiﬁcantly between the STZ+ISO control and other groups. Compared with the diabetic and isoproterenol control groups, the ISO+ SNASET 25 and 50 group had less histological damage to the heart (Table 3). This indicates that herbonanoceutical from tamoenju leaf extract protects against myocardial infarction after isoproterenol has already caused it.

Table 3. Impact of herbonanoceutical derived from tamoenju leaf extract on histopathological severity of heart muscle injury in rats

Groups	Average damage score
NC	0.80±0.00^a
STZ+ISO	1.52±0.08^d
STZ+ISO+Met+At	0.96±0.04^b
STZ+ISO+SNASET 25	1.28±0.05^c
STZ+ISO+SNASET 50	1.04±0.04^b

a b

c d

Figure 5. Histopathology of rat heart using H&E staining with 40x10 magnification (a) NC showing damage found < 25% (b) STZ+ISO showing damage found >25-50% (c) STZ+ISO+Met+At showing damage found < 25%-50% (d) STZ+ISO+SNASET 25 showing damage found < 25% (e) STZ+ISO+SNASET 50 showing damage found < 25%

: pyknosis nucleus

: muscle fibers shrink

: the distance between the fibers becomes sparse

The occurrence of acute myocardial infarction following STZ and isoproterenol administration was confirmed by microscopic examination of the tissue using HE staining, as shows in Fig. 5. Necrosis in heart cells is usually characterized by the presence of a heart cell nucleus that appears shrunken, has irregular borders and is dark in color, which is a characteristic of a pyknotic nucleus²⁷^,²⁸. In the group of rats given isoproterenol, myopathy was observed, characterized by myocardial necrosis marked by pyknotic nuclei (karyopyknosis) in heart muscle cells, shrinking of muscle fibers, increased spacing between muscle fibers, and neutrophil infiltration.

CONCLUSION:

Based on the findings of this study, the herbonanoceutical from Hibiscus surattensis L. leaf extract (HSL) demonstrates promising potential as both a cardioprotective and glucose-lowering agent, particularly at a dose of 50 mg/kg BW. The extract has been shown to protect against isoproterenol-induced heart damage in diabetic animals, as evidenced by the reduction of heart biomarkers (CK, CK-MB, LDH, SGOT, SGPT) approaching levels observed in healthy controls. Therefore, the herbonanoceutical HSL could be a promising adjuvant therapy to treat diabetes-related complications and other associated conditions

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are grateful to the Directorate General of Higher Education, Research, and Technology, Ministry of Education, Culture, Research, and Technology, for providing research grants to lecturers through the fundamental regular research programs with contract number DIPA-023.17.1.690523/2024 1st Revision, 4 February 2024, which enabled the authors to successfully conduct this research.

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Received on 28.12.2024 Revised on 13.06.2025

Accepted on 15.10.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):338-345.

DOI: 10.52711/0974-360X.2026.00049

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