PIP-SLNs were subjected to centrifugation at 4500 rpm at room temperature for 15 min. After centrifugation, the sediment was collected and dried at a temperature of 40 °C. The formulation yield (%) was calculated using the following equation (Vivek et al., 2007): %  Yield = Weight   of   SLNs   obtained Weight   of   solid   materials   used   in   the   formulation x   100

The entrapment efficiency of PIP-SLNs was estimated by dissolving freeze dried PIP-SLNs in methanol/water mixture (80:20 v/v%). The mixture was passed through the 0.4 µm nylon syringe filter, diluted appropriately with the mobile phase, and sonicated for 10 min before being analysed by the HPLC instrument (Shimadzu Prominence-I, LC-2030C 3D Plus, Tokyo, Japan). Briefly, 10 µL sample solution was injected into C18 column (Phenomenex, 5 µ size, 150 mm length, 4.6 mm internal diameter) and the flow rate was maintained to1 mL/min. The samples were analysed at 343 nm using UV spectrophotometer photodiode detector. The entrapment efficiency (%) was calculated using the following formula (Gopalakrishna et al., 2023): %  Entrapment efficiency = Amount   of   PIP   in   SLNs Initial   amount   of   PIP   added x   100

The diameter, polydispersity index (PDI) and zeta potential of the PIP-SLNs was measured using a zeta sizer (Malvern Model- Nano ZS-90, Worcestershire, United Kingdom). 10 μL of each formulation was dispersed in 10 mL of deionized water, and sonicated for 10 min. The sonicated sample was collected in a cuvette and processed for size distribution measurement. The zeta potential of the PIP-SLNs was determined using an electrode cuvette. All measurements were conducted in triplicates.

The surface morphology of the optimized formulation (F6) was studied by SEM analysis (Singh et al., 2022). The optimized formulation (F6) was photographed by scanning electron microscope (ZEISS ULTRA 55 Field emission scanning electron) at 100.00 KX magnification at 25 °C.

Swiss albino male mice (25–30 g) were used in this study. All animals were housed under the temperature conditions of 21 °C–25 °C, and RH 60% ± 10% with a 12 h/12 h light/dark cycle. The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Acharya and BM Reddy College of Pharmacy (Registration number: 997/C/06/CPCSEA). The study was performed in accordance with IAEC (IAEC/ABMRCP/2021- 22/2).

The anti-hyperlipidemic activity of the optimized formulation (F6) was evaluated in comparison with pure piperine (PIP) and the reference standard silymarin. Animals were assigned to experimental groups using simple randomization to minimize selection bias. A total of five groups were established, each containing six animals (sample size determined based on previous studies reporting effect sizes for HFD-induced hyperlipidemia models and powered to detect a minimum difference of 20%–25% in lipid profile parameters with 80% power at α = 0.05). One group served as the normal control, and the remaining four groups received disease induction and/or treatment.

The normal control group received a standard laboratory diet, while all other groups were fed a high-fat diet (HFD) composed of regular chow supplemented with 5% cholesterol and 1% cholic acid, as described in earlier reports (Sampathkumar et al., 2011; Venkateshan et al., 2016). The study duration was 21 days, and treatment was administered once daily as per the following protocol:

Group 1 - Normal Control: Normal saline solution (per oral) + regular diet

Throughout the study, animals were housed under identical environmental conditions, and treatment order was randomized daily to avoid handling-related bias. All outcome assessments were performed by investigators blinded to group allocation to ensure objective evaluation.

The impact of treatments on glucose metabolism was estimated by oral glucose tolerance test (OGTT) (Andrikopoulos et al., 2008). Briefly, mice were deprived from food for overnight. Then the mice were randomly assigned to five groups: a normal control, a disease control, and test groups receiving silymarin, pure PIP, and an optimized formula (F6) of PIP-SLNs, respectively, as aforementioned. At 30 min post each treatment, 2 g/kg glucose solution was given orally, and blood glucose levels were measured using a glucometer prior to treatments (0 min), as well as 30 min and 60 min post glucose solution intake.

After 3 days of washout period post oral glucose tolerance test, blood specimens were collected via retro-orbital sinus puncture. This was followed by centrifugation at 4800 rpm for 30 min. The obtained serum was separated and stored at −20 °C. This serum sample was used for biochemical analysis of lipid profile (cholesterol and triglycerides) and liver function enzymes (alanine aminotransferase (ALT), and aspartate aminotransferase (AST) (Fabbrini et al., 2010), using the standard diagnostic kits.

At the end of the experiments and after blood sampling, animals were euthanized using diethyl ether and the liver was dissected. For histopathological examination, liver samples were first washed by PBS, then fixed in 10% formalin, dehydrated in ascending grades of alcohol, and paraffin embedding procedures were conducted. 5 μm thickness sections were then stained by haematoxylin-eosin (H & E) dyes and the sections were screened for signs of liver injury such as vesicular steatosis, lobular inflammation (lymphocytes, Kupffer cells) ballooning, Mallory-Denk bodies, fibrosis, and necrosis.

The optimized PIP-SLNs (F6) demonstrated an average particle size of 191.2 ± 27.9 nm (Figure 2a), and the PDI was found to be 0.28. The zeta potential of the optimized formulation was measured at −20.2 ± 1.3 mV (Figure 2b), reflecting the surface charge and colloidal stability of the nanoparticles. The surface morphology of the optimized PIP-SLNs (F6) when examined using scanning electron microscopy (SEM) revealed spherical particles with smooth surfaces, although some degree of agglomeration was observed among the nanoparticles (Figure 2c).

The in vitro release study showed that PIP-SLNs (F6) exhibited a biphasic release pattern, with approximately 18% of Piperine released within the first 4 h and a cumulative release of about 70% over 48 h (Figure 3). In contrast, the piperine suspension released nearly 85% of the drug within the first 2 h.

Kinetic analysis demonstrated that the release from PIP-SLNs corresponded closely with the Higuchi model (R² = 0.976), followed by Zero-order kinetics (R² = 0.877). The Korsmeyer-Peppas model yielded an exponent of n = 0.63 (R² = 0.95). For the Piperine suspension, the release data showed the highest correlation with the First-order model (R² = 0.969), while correlations with Zero-order, Higuchi, and Korsmeyer-Peppas models were comparatively low.

The oral glucose tolerance test (OGTT) was performed to understand the impact of PIP-SLNs on hepatic glucose metabolism. The glucose tolerance profiles are presented in Figure 4. The HFD-fed mice (disease control) exhibited significantly elevated blood glucose levels at all time points compared to the normal control group (p < 0.025).

Treatment with pure PIP (group 4) and silymarin (group 3) resulted in a significant reduction in blood glucose levels, compared to the disease control. Notably, mice treated with the optimized PIP-SLNs formulation (F6) showed a substantial decline in glucose levels by the end of 60 min. Glucose utilization in the formulation treated group (F6) was closely similar to silymarin (P < 0.05) and better than pure PIP (P < 0.08), indicating the improved therapeutic potential of PIP-SLNs. These findings suggest higher glucose utilization in the F6 treated mice.

The anti-hyperlipidemic potential of the optimized PIP-SLNs (F6) was evaluated on a high-fat diet (HFD) induced hyperlipidemia mice model (Du et al., 2020). Acute exposure to HFD, significantly elevated serum total cholesterol (TC) and triglyceride (TG) levels compared to the normal control group (p < 0.025) (Figure 5a).

Treatment with the optimized PIP-SLNs (F6) effectively reduced both TC and TG levels, restoring them to near-normal values. The lipid-lowering activity of F6 was on par with the standard silymarin treatment (p < 0.05), and it outperformed pure piperine in restoring lipid levels (p < 0.102 vs. F6). (Figure 5a).

In addition, liver weight significantly increased in HFD fed mice, compared to the controls (Figure 5b). Treatment with pure PIP showed minimal improvement, while the optimized SLNs formulation (F6) significantly reduced liver weight to near normal levels. The hepatoprotective effect of F6 was observed to be equivalent to that of silymarin (p < 0.028) (Figure 5b).

The hepatoprotective activity of the optimized PIP-SLNs (F6) was assessed by determining the serum levels of liver function biomarkers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). As shown in Figure 6, the disease control group (HFD-fed mice) exhibited a significant elevation in ALT and AST levels, compared to the normal control group (p < 0.001), indicating hepatic injury.

Treatment of disease control group with pure PIP or silymarin significantly reduced serum ALT and AST levels (p < 0.001 vs. disease control). Notably, the group treated with optimized PIP-SLNs (F6) showed marked improvement, with enzyme levels nearly returning to those of the normal control. This reduction was statistically significant compared to the disease group (p < 0.001), and also equivalent to the silymarin group (p < 0.035) and better compared to pure piperine group (p < 0.026).

The protective effects of PIP-SLNs on hepatic architecture and liver structural integrity were evaluated through histopathological analysis (Figure 7). In the normal control group (Figure 7a), liver sections displayed intact parenchymal architecture with well-preserved hepatocytes and sinusoids. In contrast, the disease control group (Figure 7b) showed severe hepatocellular damage, characterized by diffuse cytoplasmic vacuolation of hepatocytes and congested blood vessels. The pure PIP treated group (Figure 7c) exhibited moderate hepatocellular degeneration, including cytoplasmic vacuolation and blood vessel congestion. The silymarin-treated group (Figure 7d) demonstrated largely preserved hepatic structure with intact hepatocytes and sinusoids. Notably, the group treated with the optimized PIP-SLNs (F6) (Figure 7e) exhibited mild hepatocellular alterations, with mostly intact sinusoids and only a few congested vessels.

Drug-excipients compatibility is essential for ensuring formulation stability and promoting efficient drug incorporation, while minimizing the risk of drug degradation or loss of bioactivity during processing and storage. Herein, FTIR analysis provided clear evidence supporting the compatibility of the selected lipid excipients with PIP and their suitability for formulating stable SLNs. The characteristic absorption bands of PIP, including the aliphatic C–H stretching (2939-2860 cm⁻¹), C=O stretching (∼1583 cm⁻¹), the C=C stretching (∼1449 cm⁻¹), and the C–O stretching (∼1253 cm⁻¹), C-H bending vibrations of the aromatic (841 cm⁻¹) and substituted aromatic ring (717 cm⁻¹) were distinctly preserved in the spectra of physical mixtures containing different lipids. The retention of these signature peaks without marked shifts, alterations in intensity, disappearance of original bands, or formation of new peaks suggests the absence of chemical incompatibilities or strong intermolecular interactions such as covalent bonding, esterification, or oxidative degradation.

The physicochemical characteristics of the F6 formulation, suggested that it was well-suited for hepatic delivery. The particle size of approximately 191 nm lied within the optimal range for passive targeting of the liver, allowing efficient passage through fenestrated hepatic capillaries (Abu Lila et al., 2024). This nano-size also supported enhanced bioavailability and improved systemic circulation. The PDI value of 0.28 indicated a uniform particle size distribution. Since PDI values below 0.3 are generally considered acceptable for colloidal stability, this result reflected the formulation’s homogeneity, and reproducibility during processing.

Zeta potential plays a vital role in predicting the stability of nanosuspensions. Nanoparticles with high surface charge-whether positive or negative, tend to repel one another, thereby preventing aggregation (Abu Lila et al., 2024). Zeta potential of −20.2 ± 1.3 mV was observed for F6 (Figure 2b) which suggested moderate electrostatic repulsion among particles and this, when combined with low PDI, contributed to the physical stability of the formulation. Although higher absolute zeta potential values (e.g., >30 mV) are generally associated with enhanced stability, a zeta potential of −20.2 ± 1.3 mV, particularly in conjunction with a stabilizing emulsifier such as poloxamer 407, can be sufficient to maintain colloidal dispersion over time.

SEM analysis provided valuable insights into the topographical characteristics of the optimized PIP-SLNs. The spherical shape and smooth surface morphology of the particles suggested successful formulation using the hot homogenization technique (Figure 2c). Generally spherical nanoparticles are preferred for drug delivery applications, as they are associated with better cellular uptake, and uniform drug distribution. The agglomeration of the particles in the lyophilized sample is not uncommon and may be attributed to the absence of a cryoprotectant during the freeze-drying process or residual moisture content (Almalik et al., 2017). However, such agglomeration typically does not impact re-dispersibility or drug release if the formulation readily reconstitutes in aqueous media. The overall morphology supported the previously observed favorable particle size and homogeneity (PDI), affirming the quality and integrity of the optimized SLNs. Overall, these findings confirmed that the optimized PIP-SLNs possess favorable characteristics, for further in vivo and pharmacological evaluation.

The OGTT results demonstrate the potential of PIP-SLNs to significantly improve glucose utilization and regulate blood glucose levels in high-fat diet-induced hyperglycemic mice. Elevated glucose levels in the HFD group confirm impaired glucose metabolism, a common manifestation of metabolic stress and insulin resistance. The glucose-lowering effect observed in the F6treated group was not only statistically significant but also biologically relevant, as it closely approached the glucose levels of the normal control group across all tested time points. This effect was superior to that of pure PIP, and equivalent to silymarin, indicating that the nanoparticle-based formulation enhanced the bioavailability, and the therapeutic efficacy of PIP.

The observed outcomes may be attributed to improved hepatic targeting and controlled release characteristics of SLNs, which potentially enhanced drug accumulation in the liver, the primary site for glucose uptake and metabolism. The results also affirmed the role of SLNs as effective delivery carriers for hydrophobic bioactive like PIP, enabling sustained action and better therapeutic outcomes in glucose regulation. Thus, the findings collectively supported the potential of optimized PIP-SLNs as a promising formulation approach for managing glucose dysregulation in metabolic disorders, such as diabetes and non-alcoholic fatty liver disease.

Excessive fat accumulation from high-fat dietary intake led to dyslipidemia and hepatic steatosis, hallmark features of metabolic disorders such as non-alcoholic fatty liver disease (NAFLD) (Elnaggar et al., 2015). The current study demonstrated the potent anti-hyperlipidemic and hepatoprotective effects of the optimized PIP-SLNs (F6) in an HFD-mice model. The significant reduction in serum TC and TG levels following F6 treatment, implied enhanced lipid-lowering activity, which was comparable to the standard silymarin and superior to pure PIP (Figure 5a). These findings favored the notion that encapsulation of PIP in SLNs enhanced its bioavailability, stability, and therapeutic efficacy, likely through improved hepatic delivery and prolonged circulation. Additionally, the ability of F6 to restore liver weight to normal levels suggested that it protects against hepatic lipid accumulation and structural damage. Interestingly, this effect surpassed that of silymarin, highlighting the enhanced hepatoprotective potential of the nano-formulated PIP (Figure 5b). The superior performance of F6 over pure PIP also underlines the benefit of SLNs in overcoming the limitations of hydrophobic drugs, such as poor solubility and rapid metabolism. Collectively, these results reinforced the therapeutic usage of PIP-SLNs based delivery systems, in managing HFD induced metabolic dysfunctions, including hyperlipidemia and fatty liver-associated hepatomegaly.

Serum aminotransferases: aspartate aminotransferase (AST), and alanine aminotransferase (ALT), are essential enzymes that aid in carbohydrate and amino acid metabolism (Fabbrini et al., 2010). Elevated aminotransferases, particularly ALT and AST, are hallmark indicators of hepatocellular damage in metabolic liver diseases such as in NAFLD. The marked rise in these enzymes in HFD-fed mice confirms the onset of hepatic dysfunction in this model.

Treatment with pure PIP or silymarin moderately reduced enzyme levels, reflecting some degree of hepatoprotection. However, the optimized PIP-SLNs (F6) demonstrated a notable protective effect, efficiently restoring ALT and AST levels to near normal values (Figure 6). This pronounced effect highlighted the potential of SLNs in enhancing drug delivery to hepatic tissues (Mishra et al., 2018), likely due to improved solubility, bioavailability, and sustained release of PIP.

The significant difference in efficacy between F6 and both pure PIP and silymarin further reinforced the hypothesis that nanoencapsulation improved the therapeutic outcomes, especially in conditions like NAFLD that require targeted hepatic intervention (Ashfaq et al., 2023; Guo et al., 2016). These findings confirmed the role of PIP-SLNs (F6) as a promising hepatoprotective agent in the context of diet-induced liver injury.

Histopathological examination provided critical insight into the tissue level damage, and recovery associated with liver injury. In this study, the high fat diet (HFD) induced marked hepatic damage, evident from diffuse hepatocyte vacuolation, and vascular congestion, in the disease control group (Baz et al., 2022); thus, reflecting the pathological features of non-alcoholic fatty liver disease (NAFLD). Treatment with pure PIP showed only partial protection, as evidenced by moderate hepatocellular injury. Conversely, the silymarin-treated group exhibited considerable protection with largely preserved hepatic architecture, supporting its established role as a hepatoprotective agent. Importantly, animals treated with the optimized PIP-SLNs (F6) demonstrated a substantial histological improvement. The presence of only mild hepatocyte vacuolation, and largely intact sinusoids suggested effective hepatoprotection (Yang et al., 2017). These findings indicated that the SLN-based delivery system enhanced hepatic accumulation of PIP, contributing to improved anti-hyperlipidemic and hepatoprotective outcomes compared to pure drug administration.

Comparable outcomes have been reported for curcumin and resveratrol based nano therapies. Curcumin nanoparticles have been shown to lessen hepatic steatosis and inflammatory responses by improving bioavailability and directing drug delivery to target tissues (Jazayeri-Tehrani et al., 2019). Likewise, nano-resveratrol systems have demonstrated benefits by mitigating oxidative stress and modulating lipid metabolism through sustained release and enhanced hepatic uptake (Teng et al., 2019). In this context, the notable hepatoprotective effects achieved with PIP-SLNs such as decreased steatosis, reduced vacuolar changes, and improved biochemical markers indicate that nano-encapsulated piperine (PIP-SLNs) may offer therapeutic benefits comparable to these established nano-formulations, underscoring the need for future direct comparative evaluations.