Antidiabetic and Histoprotective Effects of Microemulsion Formulation of Red Gedi Leaf (Abelmoschus manihot) Extract in Streptozotocin-Induced Rats

The condition known as Diabetes Mellitus has expanded into a widespread global disturbance rather than a contained medical issue. Within this landscape, Indonesia holds a high position, with a prevalence of about 8.7% and a projected surge toward 19.5 million individuals by 2045, according to the International Diabetes Federation [1]. At a narrower scale in Central Sulawesi, records from 2021 illustrate an imbalance where Palu documented 23,677 cases while only 1,314 people, or 5.5%, accessed adequate treatment services [2]. This imbalance signals a structural disconnect that calls for practical and reachable therapeutic options.

The pathophysiology of diabetes mellitus is characterized by chronic hyperglycemia resulting from either insufficient insulin production by pancreatic β cells or impaired insulin-mediated glucose uptake by peripheral tissues. An unhealthy lifestyle and sedentary behavior contribute to the development of insulin resistance, initiating a cascade of metabolic dysfunction [3]. The persistent hyperglycemic state triggers excessive production of reactive oxygen species (ROS), including 8-hydroxy-deoxyguanosine and malondialdehyde, which induce oxidative damage to cellular macromolecules [4]. This oxidative stress directly compromises the integrity of deoxyribonucleic acid (DNA) structure, leading to progressive cellular dysfunction and apoptosis of pancreatic β cells [5]. Furthermore, ROS accumulation exacerbates insulin resistance in target tissues by interfering with insulin receptor signaling pathways, creating a vicious cycle of metabolic deterioration [6]. The systemic effects of uncontrolled diabetes extend beyond glycemic dysregulation, causing progressive damage to multiple organs, including the stomach, liver, and kidneys, through oxidative injury and microvascular complications [3].

Despite advances in diabetes management, current therapeutic options present several limitations. Synthetic antidiabetic drugs, while effective in glycemic control, are associated with adverse effects, accessibility challenges, and high treatment costs that burden patients, particularly in resource-limited settings [7, 8]. Glibenclamide, a commonly prescribed sulfonylurea, effectively stimulates insulin secretion from pancreatic β cells [9], but long-term use may lead to β cell exhaustion, hypoglycemic episodes, and secondary failure [10]. These limitations highlight the necessity for alternative therapeutic strategies that offer efficacy with improved safety profiles and better accessibility.

Traditional medicinal plants offer promising alternatives for diabetes management, with many species demonstrating significant antidiabetic properties through diverse phytochemical constituents [11, 12]. In Central Sulawesi, red gedi leaf (Abelmoschus manihot (L.) Medik.) has been traditionally utilized for treating various ailments, including canker sores, diabetes mellitus, ulcers, kidney disease, hypercholesterolemia, and facilitating childbirth. Phytochemical analysis has identified key bioactive compounds in red gedi leaves, including flavonoids, alkaloids, saponins, and tannins, which collectively contribute to its antidiabetic properties [13]. These compounds exert their therapeutic effects through multiple mechanisms: flavonoids enhance insulin sensitivity and reduce oxidative stress, alkaloids modulate glucose metabolism, saponins improve lipid profiles, and tannins provide antioxidant protection against ROS-induced cellular damage [14].

Previous investigations have demonstrated the dose-dependent efficacy of red gedi leaf extract in diabetic animal models. A study revealed that 300 mg/kg BW extract resulted in a significant decrease in blood glucose levels and effectively enhanced pancreatic regeneration, while 450 mg/kg BW substantially increased insulin levels [15]. Further research confirmed that 300 mg/kg BW administration successfully reduced blood glucose, malondialdehyde, and 8-hydroxy-deoxyguanosine levels while stimulating insulin production in streptozotocin-induced diabetic rats. However, these relatively high doses present practical challenges, including potential adverse effects, increased treatment costs, and reduced patient compliance [16, 17].

Conventional herbal extract preparations face significant pharmaceutical challenges, particularly regarding bioavailability and therapeutic efficacy. Many phytochemical compounds, especially flavonoids and polyphenols present in red gedi leaves, exhibit poor aqueous solubility and limited membrane permeability, resulting in suboptimal absorption and reduced therapeutic outcomes [11, 12]. Microemulsion technology offers an innovative solution to overcome these limitations by creating thermodynamically stable, optically transparent dispersions with nanoscale droplet sizes (typically 20-200 nm) [18, 19].

Microemulsions significantly enhance the solubility and bioavailability of poorly water-soluble phytochemicals such as flavonoids, enabling superior absorption across biological membranes and improved pharmacological efficacy [20, 21]. The nanoscale particle size facilitates enhanced cellular uptake and tissue penetration, allowing active compounds to reach specific target receptors more efficiently [22]. Additionally, microemulsion systems provide protective encapsulation of bioactive compounds, preventing degradation in the gastrointestinal environment and maintaining stability during storage [23, 24]. This formulation strategy enables dose reduction while maintaining or enhancing therapeutic effects, thereby minimizing potential adverse reactions and improving patient compliance [7, 16]. The spontaneous formation and thermodynamic stability of microemulsions ensure consistent drug delivery and reproducible therapeutic outcomes [25].

While previous studies have established the antidiabetic potential of red gedi leaf extract at doses of 300–450 mg/kg BW [15], there remains a critical knowledge gap regarding the efficacy of lower doses when formulated as microemulsions. The novelty of this research lies in the application of microemulsion technology to substantially reduce the required therapeutic dose of red gedi leaf extract while simultaneously maintaining or enhancing efficacy. This approach has not been previously investigated for Abelmoschus manihot extract in the context of diabetes management and multi-organ protection. The three dose levels tested in this study (25, 50, and 100 mg/kg BW) were selected based on a proportional dose-reduction strategy derived from the previously established effective doses of 300–450 mg/kg BW for conventional extract [15]; these represent approximately 8–33% of the conventional effective dose range, thereby enabling a systematic exploration of dose-response relationships within a therapeutically meaningful and practically feasible range. The upper limit of 100 mg/kg BW was determined to avoid potential toxicity risks while still allowing assessment of an intermediate dose (50 mg/kg BW) and a sub-therapeutic starting point (25 mg/kg BW). Future studies should consider extending the dose range both below 25 mg/kg BW and above 100 mg/kg BW to fully characterize the dose-response curve and determine the minimum effective and maximum tolerated doses of the microemulsion formulation. Furthermore, the mechanism by which microemulsion enhances bioavailability is primarily attributable to its nanoscale droplet size (20–200 nm), which increases the surface area available for gastrointestinal absorption, reduces the diffusion layer thickness at the mucosal interface, and promotes enhanced transcellular and paracellular transport of encapsulated phytochemicals. This results in higher plasma concentrations and improved tissue-level distribution compared to conventional extract preparations [18, 20, 21].

Therefore, this study aimed to evaluate the antidiabetic efficacy of red gedi leaf ethanol extract-based microemulsion at various doses (25, 50, and 100 mg/kg BW) on blood glucose levels and histopathological profiles of major metabolic organs (pancreas, liver, kidneys, and stomach) in streptozotocin-induced diabetic rats. By demonstrating that significantly lower doses formulated as microemulsions can achieve therapeutic effects comparable to or superior to conventional higher-dose extracts, this research contributes to the development of more cost-effective, safer, and patient-friendly herbal antidiabetic formulations.

Eight formulations combining VCO, Tween 80, and glycerin were evaluated (Figure 1). Formulation 6 consisted of Tween 80 (35 mL), glycerin (30 mL), VCO (20 mL), and distilled water (15 mL) — in a ratio of 35:30:20:15. Among the eight formulations, Formulation 6 and Formulation 7 both demonstrated visual clarity and physical stability throughout eight weeks of observation, while the remaining six formulations exhibited turbidity and phase separation. Formulation 6 was ultimately selected as the base formula, as both formulations yielded comparable results. The selected base formula contained 35 mL of Tween 80, 30 mL of glycerin, 20 mL of VCO, and 15 mL of distilled water (total 100 mL). Three dose-specific formulations (Table 1(a, b, c)) were then prepared by dissolving 200 mg (for 25 mg/kg BW), 400 mg (for 50 mg/kg BW), or 800 mg (for 100 mg/kg BW) of red gedi leaf ethanol extract into this identical base, yielding stock concentrations of 2, 4, and 8 mg/mL, respectively. A surfactant-to-co-surfactant ratio of 1.17:1 and a total surfactant concentration of 65% supported spontaneous formation and long-term stability [27]. Encapsulation efficiency of the three microemulsion formulations was 89.4 ± 1.2%, 91.2 ± 0.9%, and 92.6 ± 1.1% for the 25, 50, and 100 mg/kg BW dose formulations, respectively, indicating highly efficient entrapment of the extract within the micellar core. Drug loading values were 0.20 ± 0.01%, 0.39 ± 0.01%, and 0.75 ± 0.02% for the respective formulations. In vitro release profiling demonstrated a biphasic pattern: an initial burst release of 30–35% within the first 2 hours, followed by sustained release reaching approximately 80–85% cumulative release at 24 hours. This prolonged release profile supports the once-daily oral dosing regimen employed and is consistent with the enhanced pharmacological efficacy observed at lower doses compared to conventional extract preparations [25, 27].

Figure 1. Microemulsion stability results of red gedi leaf ethanol extract

Table 1a. Microemulsion formulation composition for dose 25 mg/kg BW (stock concentration: 2 mg/mL)

Notes: VCO: Virgin Coconut Oil

Table 1b. Microemulsion formulation composition for dose 50 mg/kg BW (stock concentration: 4 mg/mL)

Notes: VCO: Virgin Coconut Oil

Table 1c. Microemulsion formulation composition for dose 100 mg/kg BW (stock concentration: 8 mg/mL)

Notes: VCO: Virgin Coconut Oil

Phytochemical screening (Tables 2-3) confirmed alkaloids, flavonoids, saponins, and tannins. Quantitative analysis revealed 22.48% flavonoid (quercetin equivalent), 2.70% saponin, 1.34% tannin, and 0.33% alkaloid (quinine equivalent).

Table 2. Qualitative test results of red gedi leaf extract microemulsion

Table 3. Quantitative test results of red gedi leaf extract microemulsion

Physicochemical characterization (Table 4) showed particle size of 157.3 nm, polydispersity index of 0.430, zeta potential of -28.5 ± 2.1 mV, conductivity of 145.7 ± 3.8 μS/cm, viscosity of 48.3 ± 1.5 cP, and pH of 6.2 ± 0.3.

Table 4. Physicochemical characterization parameters of red gedi leaf ethanol extract microemulsion

Glucose measurements (Table 5 and Figure 2) on day 0 ranged from 50 to 135 mg per dL in all groups, indicating similar baseline conditions. By day 7, glucose levels increased in all groups except the normal group, confirming successful induction of diabetes. On day 14, untreated diabetic rats showed higher glucose levels compared to the treated groups. By day 21, the 100 mg per kg body weight microemulsion produced a stronger glucose-lowering effect than Glibenclamide. On day 28, both the positive control and the 100 mg per kg group differed significantly from the untreated group, with the highest dose showing the greatest reduction of about 91.80 mg per dL, outperforming higher dose extract comparisons reported elsewhere [20].

Table 5. Average blood glucose level results

Histopathological analysis (Table 6 and Figure 3) showed that the negative control had the highest score (4) as a reference for damage, while the normal control served as a healthy baseline. The 100 mg/kg BW microemulsion showed the most significant protection, showing only mild degeneration (score 1.2) compared to the lower dose groups (scores 2.6–2.8). This superior protection at the 100 mg/kg BW dose may be due to the higher concentration of flavonoids and tannins reaching the pancreatic tissue, which collectively suppress ROS-mediated β-cell apoptosis and attenuate the NF-κB inflammatory signaling pathway triggered by STZ-induced oxidative stress. The nano-droplet size (157.3 nm) of the microemulsion likely facilitated increased penetration of these bioactives into the pancreatic microvascular network, allowing for more efficient cellular uptake and tissue repair compared to lower doses. Figure 4 shows severe pancreatic necrosis in the negative control (score 4), while the positive control and treated groups showed progressively reduced damage. The positive control and the 25 mg/kg BW dose both showed significant differences compared to the negative control, indicating a marked improvement in pancreatic tissue. The 50 and 100 mg/kg BW doses differed significantly from the normal control, with low scores approaching normal. Overall, all treatment groups successfully improved pancreatic histopathology compared to the negative control, with the 100 mg/kg BW dose providing the best results, closest to normal tissue.

Table 6. Histopathological scoring of the pancreas of male white rats

When the gastric layer was inspected in male albino Rattus norvegicus, the contrast between groups unfolded almost like a spectrum of structural collapse. The untouched cohort sat at a perfect stillness of 0 ± 0.000 (Table 7), a microscopic landscape without visible disruption (Figure 5(A)). In sharp opposition, the diabetic untreated group climbed to 3 ± 1.225 (Table 7), where more than half of the cellular terrain, roughly 51–75%, had disintegrated into necrotic debris (Figure 5(B)). The comparator receiving Glibenclamide settled at 2.4 ± 0.894 (Table 7), where 21–50% of cells were already lost, and inflammatory infiltration appeared dense (Figure 5(C). Parallel patterns persisted in the 25 and 50 mg/kg BW groups, registering 2.4 ± 0.548 and 2.2 ± 0.837 (Table 7), with necrotic zones still occupying 21–50% and inflammatory cells scattered heavily (Figure 5(D)–(E)). Only the 100 mg/kg BW intervention seemed to rewrite the script, dropping the score to 1.2 ± 0.447 (Table 7), where destruction shrank dramatically to just 1–20% of cells (Figure 5(F)).

Table 7. Histopathological scoring of the stomach of male white rats

In Figure 3, the negative control recorded the highest score (4) as a reference for stomach damage. The positive controls, doses of 25 and 50 mg/kg BW, did not show any statistically significant differences. Only the 100 mg/kg BW dose showed a significant difference compared to the negative control and the normal control, making it the only treatment group with significant improvement in gastric histopathology.

Liver histopathology results can be seen in Table 8. The normal control group showed intact hepatocyte architecture with no signs of damage (Figure 6(A)). The negative control exhibited severe hepatocyte necrosis and extensive inflammatory infiltration (Figure 6(B)), while the positive control (glibenclamide) showed moderate improvement (Figure 6(C)). The 25 mg/kg BW microemulsion produced an average damage score of 2.4 with 51–75% hepatocyte degeneration (Figure 6(D)). The 50 mg/kg BW treatment produced a score of 1.6 with 21–50% damage (Figure 6(E)), while the 100 mg/kg BW dose achieved the smallest score, namely 1.2, indicating only mild degeneration affecting 1–20% of hepatocytes (Figure 6(F)).

Table 8. Histopathological scoring of the liver of male white rats

Histological features were inconsistently distributed across the liver sections. Figure 3 positions the untreated diabetic group at the extreme, reaching a maximal injury score of 4, marking extensive hepatic disruption. Introducing 25 and 50 mg/kg BW began to soften that damage, with both groups diverging significantly from the untreated condition, and the 50 mg/kg BW dose showing a more noticeable structural recovery. The comparator group and the 100 mg/kg BW dose both moved closer to physiological normality, although still statistically distinct from the untouched baseline. Among all interventions, the 100 mg/kg BW dose produced hepatic tissue that most closely resembled the intact state, as if the organ had partially retraced its path back to normalcy.

Kidney histopathology analysis (Table 9) revealed mean damage scores of 2.6, 2.0, and 1.6 for doses of 25, 50, and 100 mg/kg BW, respectively. The normal control group showed intact glomerular and tubular architecture with no cellular damage (Figure 7(A)). The negative control exhibited severe glomerular damage and tubular necrosis affecting more than 75% of the renal tissue (Figure 7(B)). The positive control group treated with glibenclamide showed moderate improvement with reduced necrosis (Figure 7(C)). The 25 mg/kg BW dose showed moderate tubular degeneration affecting 51–75% of the area (Figure 7(D)), while the 50 mg/kg BW dose showed mild-to-moderate damage affecting 21–50% of the renal tissue (Figure 7(E)). The 100 mg/kg BW dose produced only mild tubular necrosis affecting 1–20% of the illustrated area (Figure 7(F)).

Table 9. Histopathological scoring of the kidney of male white rats

In Figure 3, the negative control obtained the highest score (4) as a measure of gastric damage. The positive controls, at doses of 25 and 50 mg/kg BW, showed no statistically significant differences. Only the 100 mg/kg BW dose showed a significant difference compared to both the negative and normal controls, making it the only treatment group with a significant improvement in gastric histopathology.

Across the entire organ panel, a recurring pattern emerged. The 100 mg/kg BW microemulsion consistently occupied the lowest damage interval, ranging from 1.2 to 1.6. This range stood in clear separation from the untreated diabetic condition, which hovered between 3.0 and 4.0 with statistical significance at p < 0.05. In several instances, this high dose not only paralleled but subtly surpassed the comparator benchmark, suggesting a system-wide protective effect rather than a localized one.

Among all tested conditions, the 100 mg/kg BW microemulsion derived from Abelmoschus manihot leaves produced the most pronounced decline in blood glucose within diabetic Rattus norvegicus. Beyond glycemic control, it moderated structural injury across pancreatic islets of Langerhans, hepatocytes, renal filtration units, and gastric tissue layers. This suggests that microemulsion-based delivery amplifies both the availability and the functional impact of the plant extract in addressing diabetic complications. The effectiveness at a substantially lower dose compared to conventional extract ranges of 300–450 mg/kg BW hints at improved adherence potential and cost efficiency. Additionally, the system’s spontaneous formation and thermodynamic stability support manufacturability without complex infrastructure, strengthening its feasibility as an accessible herbal therapeutic. Nevertheless, extended in vivo safety profiling remains essential, including long-term hepatic, renal, and gastrointestinal evaluations, alongside mechanistic studies, pharmacokinetic mapping in humans, storage stability testing, and economic feasibility comparisons with traditional formulations.