Nanoemulsion of Coleus Scutellarioides Leaf Extract Ameliorates Diabetes-Induced Hyperglycemia and Organ Damage in Rats

2.1 Materials

Equipment: Vacuum Rotatory Evaporator, Magnetic Stirrer (Heidolph®), Particle Size Analyzer (Horiba-SZ 100z®), Ultrasonicator (Zeta Sonic ZS-640 DB®), Transmission Electron Microscopy (TEM), UV-Vis Spectrophotometer (Single Beam®), Analytical Balance (Ohaus PA214 SKZO®), Viscometer (NDJ-8S®), pH meter, and standard laboratory glassware. 

Chemicals: Virgin Coconut Oil (VCO), Tween 80, Propylene glycol, 96% ethanol, Streptozotocin (Bioworld USA), Glibenclamide powder, and analytical grade reagents.

2.2 Plant material and extract preparation

Fresh leaves of C. scutellarioides were collected, authenticated, dried, and powdered. Maceration was performed using 96% ethanol (1:10 w/v) for 3 × 24 hours with occasional stirring. The extract was filtered and concentrated using a rotary evaporator at 50℃ under reduced pressure.

2.3 Nanoemulsion formulation

Nanoemulsions were prepared using the spontaneous emulsification method. Three formulations (F1, F2, F3) were prepared with varying ratios of VCO: Tween 80: Propylene glycol. F1 (1:10:5): VCO (1 g) was mixed with Tween 80 (10 g) and propylene glycol (5 g) containing the extract. The mixture was stirred at 1000 rpm for 30 minutes at room temperature (25±2℃), then subjected to ultrasonication at 40 kHz for 10 minutes in an ice bath to prevent temperature increase. F2 (2:10:5) and F3 (3:10:5) were prepared similarly with adjusted VCO ratios.

2.4 Nanoemulsion characterization

Particle Size Analysis (PSA): Performed using Dynamic Light Scattering (Horiba-SZ 100z®). Samples were diluted 1:100 with distilled water before measurement. Z-average diameter, PDI, and zeta potential were recorded in triplicate.

pH Measurement: Determined using a calibrated pH meter at 25°C (n = 3).

Viscosity: Measured using NDJ-8S viscometer at 25℃ with spindle speed of 60 rpm (n = 3).

Transmittance: Measured at 650 nm using a UV-Vis spectrophotometer with distilled water as a blank (n = 3).

Transmission Electron Microscope (TEM): Samples were diluted and placed on copper grids, negatively stained, and observed at various magnifications (15,000×, 43,000×, and 97,000×).

2.5 Phytochemical screening

Qualitative analysis was performed using standard methods to identify alkaloids (Dragendorff's reagent), flavonoids (Mg powder-HCl test), saponins (foam test), tannins (FeCl₃ test), and steroids (Liebermann-Burchard test).

2.6 Animal study

Thirty male Wistar rats (Rattus norvegicus), aged 2-3 months, weighing 150-200 g, were obtained from Thirty male Wistar rats (Rattus norvegicus), aged 2-3 months, weighing 150-200 g, were obtained from Gold Mice Farm Makkasar. The animals were housed in standard polypropylene cages under controlled conditions (temperature 25±2℃, 12-hour light/dark cycle) with free access to standard pellet feed and drinking water ad libitum. This study was approved by the Research Ethics Committee of the Faculty of Medicine, Tadulako University, number: 2667/UN28.10/KL/2024.

After 7 days of acclimatization, rats were randomly divided into 6 groups (n = 5 per group). Normal Control (NC): Non-diabetic rats receiving vehicle (nanoemulsion base without extract). Negative Control (STZ): Diabetic rats receiving vehicle only. Positive Control (STZ+Glib): Diabetic rats receiving glibenclamide (0.45 mg/kg BW). Treatment 1 (STZ+NE50): Diabetic rats receiving nanoemulsion at 50 mg/kg BW. Treatment 2 (STZ+NE75): Diabetic rats receiving nanoemulsion at 75 mg/kg BW. Treatment 3 (STZ+NE100): Diabetic rats receiving nanoemulsion at 100 mg/kg BW.

After overnight fasting (12 hours), diabetes was induced by a single intraperitoneal injection of freshly prepared streptozotocin (100 mg/kg BW) dissolved in 0.1 M citrate buffer (pH 4.5). Normal control received citrate buffer only. Blood glucose was measured 72 hours post-injection using a glucometer (tail vein blood). Rats with fasting blood glucose >250 mg/dL were considered diabetic and included in the study.

Starting from day 7 post-induction, treatments were administered once daily via intraperitoneal injection for 28 consecutive days. All administrations were performed between 09:00 and 11:00 AM to minimize circadian variations.

2.7 Blood glucose monitoring

Fasting blood glucose (FBG) was measured on days 0 (baseline), 7 (post-induction), 14, 21, and 28. The percentage reduction in blood glucose was calculated as:

$\%$ Reduction $=[($ FBG Day $7-$ FBG Day X $) /$ FBG Day 7$] \times 100$             (1)

2.8 Biochemical analysis

On day 28, after 12-hour fasting, rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg). Blood samples (3-5 mL) were collected via cardiac puncture into plain tubes. Serum was separated by centrifugation at 3000 rpm for 15 minutes. Urea and creatinine levels were measured using an automated clinical chemistry analyzer following standard protocols.

2.9 Histopathological examination

Following blood collection, rats were euthanized, and pancreatic tissues were immediately harvested, fixed in 10% neutral buffered formalin for 24 hours, processed, embedded in paraffin, sectioned at 5 μm thickness, and stained with hematoxylin and eosin (H&E).

Three sections per animal were examined by a certified pathologist blinded to the experimental groups. Five random high-power fields (400×) per section were evaluated. Pancreatic damage was scored based on the percentage of affected tissue showing degenerative changes and necrosis in both islets of Langerhans and exocrine tissue: Score 0: No damage/normal (0%), Score 1: Mild damage (1-20%), Score 2: Moderate damage (21-50%), Score 3: Severe damage (51-75%), Score 4: Very severe damage (>75%). The scoring system was adapted from established pancreatic injury grading systems [13-15].

2.10 Statistical analysis

Data were expressed as mean ± standard deviation (SD). Normality was assessed using the Shapiro-Wilk test. Normally distributed data were analyzed using one-way ANOVA followed by LSD or Duncan's post-hoc test. Non-normally distributed data (histopathological scores) were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test for pairwise comparisons. Statistical significance was set at p < 0.05. All analyses were performed using SPSS Statistics version 25.0.

Formulation 2 demonstrated superior physicochemical characteristics, making it the optimal formulation. The particle size of 59.2 nm falls within the ideal nanoemulsion range (20-200 nm), providing maximum surface area for enhanced bioavailability of active compounds [8, 9]. This size is crucial because particles below 100 nm can effectively penetrate the intestinal mucosa and achieve improved oral absorption. The PDI of 0.353, although slightly higher than ideal (<0.3), remains acceptable for pharmaceutical applications and indicates reasonably uniform size distribution [9].

The negative zeta potential (-32.26 mV) exceeds the threshold for electrostatic stability (±30 mV), preventing particle aggregation and ensuring long-term colloidal stability [16, 17]. The high transmittance (92.6%) confirms excellent optical clarity, correlating with small particle size and minimal light scattering–a hallmark of true nanoemulsions versus conventional emulsions.

TEM analysis revealed some non-spherical particle morphology in F2. This deviation from perfect sphericity may result from the specific surfactant-cosurfactant ratio affecting interfacial film flexibility, or rapid solvent evaporation during sample preparation [18-23]. Importantly, this morphological variation did not compromise nanoemulsion stability or biological performance, as evidenced by consistent therapeutic effects throughout the 28-day study period.

The nanoemulsion formulation addresses critical limitations of conventional C. scutellarioides extracts. The lipophilic bioactive compounds (flavonoids, terpenoids) in miana leaves have poor aqueous solubility, limiting their absorption and bioavailability [19, 20]. Nanoemulsion technology solubilizes these compounds in nano-sized oil droplets stabilized by surfactant layers, creating a thermodynamically stable delivery system that:

(1) Increases surface area: The extremely small droplet size dramatically increases the interfacial area available for absorption.

(2) Enhances permeability: Nanoparticles can penetrate through tight junctions and undergo transcellular transport across the intestinal epithelium.

(3) Protects active compounds: The oil phase shields sensitive phytochemicals from degradation in the harsh gastrointestinal environment.

(4) Improves lymphatic uptake: Lipid-based formulations promote absorption via the intestinal lymphatic system, bypassing first-pass metabolism.

This enhanced delivery explains why lower doses of nanoemulsion achieved comparable or superior effects compared to conventional extracts reported in literature [11, 13].

Our study revealed an intriguing dose-response relationship where different doses exhibited optimal efficacy for different parameters:

Blood glucose reduction (50 mg/kg optimal): The 50 mg/kg dose achieved 84.3% glucose reduction by day 28, slightly superior to higher doses, though differences were not statistically significant. This suggests a plateau effect where the 50 mg/kg dose provides sufficient active compounds to saturate insulin signaling pathways and glucose uptake mechanisms. The hypoglycemic effect likely operates through multiple mechanisms:

(1) Insulin secretagogue activity: Flavonoids in C. scutellarioides may stimulate residual β-cells to secrete insulin, similar to sulfonylureas [24].

(2) Enhanced insulin sensitivity: Bioactive compounds may activate the PI3K/Akt signaling pathway, promoting GLUT4 translocation in peripheral tissues [25, 26].

(3) Inhibition of hepatic gluconeogenesis: Alkaloids may suppress key gluconeogenic enzymes [27].

The highest dose (100 mg/kg) demonstrated superior nephroprotective effects, normalizing both creatinine and urea levels. This dose-dependent renal protection suggests that kidney damage in diabetes requires higher concentrations of bioactive compounds to:

(1) Scavenge ROS: Advanced glycation end-products (AGEs) generate excessive oxidative stress in diabetic kidneys. Higher doses provide more antioxidant flavonoids and phenolic compounds to neutralize free radicals [28, 29].

(2) Reduce inflammation: Tannins and saponins exert anti-inflammatory effects by suppressing NF-κB activation and pro-inflammatory cytokines (TNF-α, IL-6) [25].

(3) Preserve glomerular function: Prevention of mesangial matrix expansion and podocyte injury requires sustained high concentrations of protective phytochemicals.

The dose-response curve for renal protection appears steeper than for glucose control, explaining why 100 mg/kg was necessary to achieve normalization.

Interestingly, the intermediate dose (75 mg/kg) provided optimal pancreatic protection (score 2.00), better than both lower and higher doses. This "inverted U-shaped" dose-response may reflect:

(1) Optimal regenerative signaling: The 75 mg/kg dose may achieve ideal concentrations for activating pancreatic stellate cells and β-cell proliferation pathways without causing oxidative stress from excess compounds [30-32].

(2) Anti-apoptotic effects: Alkaloids at moderate concentrations can inhibit caspase activation and preserve β-cell viability, while excessive doses might have pro-oxidant effects [33, 34].

(3) Balanced angiogenesis: VEGF expression for pancreatic microvascular restoration may be optimally modulated at this dose [35, 36].

Phytochemical screening confirmed the presence of alkaloids, flavonoids, saponins, tannins, and steroids in the nanoemulsion. These diverse secondary metabolites explain the multi-organ protective effects through complementary mechanisms:

(1) Flavonoids: Act as potent antioxidants (ROS scavenging), insulin mimetics (activating insulin receptor substrate), and anti-inflammatory agents [37].

(2) Alkaloids: Modulate glucose metabolism, inhibit α-glucosidase and α-amylase (reducing postprandial glucose), and protect against STZ-induced β-cell toxicity [38].

(3) Saponins: Enhance insulin secretion, improve lipid profiles, and exhibit nephroprotective effects through anti-inflammatory actions [39].

(4) Tannins: Provide astringent effects that reduce intestinal glucose absorption and protect against oxidative kidney damage [40].

This phytochemical synergy underlies the holistic therapeutic effects observed across multiple organ systems, characteristic of herbal medicines but challenging to achieve with single-target synthetic drugs.

Previous studies on C. scutellarioides have reported antidiabetic effects, but typically used conventional extracts at higher doses (200-400 mg/kg) with less comprehensive efficacy assessments [20]. Our nanoemulsion formulation achieved:

(1) Lower effective doses: 50-100 mg/kg versus 200-400 mg/kg in conventional studies.

(2) Faster onset of action: Significant glucose reduction by day 14 versus 21-28 days.

(3) Multi-organ protection: Simultaneous pancreatic and renal benefits are rarely documented together.

(4) Superior stability: Maintained therapeutic effects over 28 days without degradation.

These advantages directly result from nanoemulsion technology, enhancing bioavailability and cellular uptake of phytochemicals. The nanoscale delivery system overcomes the bioavailability limitations that have hindered clinical translation of promising herbal medicines.

Compared to other plant-based nanoemulsions for diabetes (e.g., Gymnanthemum amygdalinum [26], Moringa oleifera [41]), our C. scutellarioides nanoemulsion showed comparable efficacy with the added advantage of robust renal protection–a critical consideration given that diabetic nephropathy is a leading cause of end-stage renal disease.

The differential dose-response relationships observed have important clinical implications. A combination therapy approach using 75 mg/kg for pancreatic protection supplemented with 100 mg/kg for renal protection might provide optimal multi-organ benefits. However, the 100 mg/kg dose appears most clinically relevant as it normalizes renal function (critical for long-term diabetes management), achieves substantial glucose reduction (81.6%), and provides adequate pancreatic protection (score 2.33). The nanoemulsion's stability and bioavailability make it suitable for oral formulation development, potentially as capsules or ready-to-drink preparations for diabetic patients. Future clinical trials should investigate:

(1) Pharmacokinetics: Absorption, distribution, metabolism, and elimination profiles.

(2) Safety profile: Long-term toxicity, drug interactions, and optimal therapeutic window.

(3) Efficacy in type 2 diabetes: Most human diabetes is type 2; insulin-resistant models needed

(4) Comparative studies: Head-to-head trials against standard antidiabetic medications.

Several limitations warrant consideration:

(1) STZ-induced model: Represents acute type 1 diabetes; may not fully reflect chronic type 2 diabetes pathophysiology in humans.

(2) Short duration: 28-day treatment period; longer studies needed to assess sustained efficacy and potential complications.

(3) Route of administration: Intraperitoneal injection used for standardization; oral bioavailability needs confirmation.

(4) Sample size: Small group sizes (n = 3-5) limit statistical power for histopathological analysis.

(5) Mechanism elucidation: Molecular pathways require investigation using Western blot, RT-PCR, and immunohistochemistry.