Nanoformulation of cerium oxide nanoparticles using pink pepper (Schinus terebinthifolius Raddi) for the management of type 2 diabetes

Technical Summary: Nanoformulation of Cerium Oxide Nanoparticles Using Schinus terebinthifolius for Type 2 Diabetes Management

Problem Statement
Type 2 diabetes (T2D) is characterized by chronic hyperglycemia, insulin resistance, and persistent inflammation, which drive oxidative stress and vascular complications. The progression of the disease involves the release of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and the generation of free radicals that damage endothelial cells and pancreatic β-cells. While cerium oxide nanoparticles (CeO₂ NPs) possess intrinsic antioxidant properties due to their Ce³⁺/Ce⁴⁺ redox cycling, and plant extracts like Schinus terebinthifolius (pink pepper) offer antioxidant and anti-inflammatory benefits, few studies have explored the synergistic potential of combining these agents. This research addresses the need for a novel nanotherapeutic strategy that leverages the enzyme-mimetic activity of nanoceria alongside the bioactive phytochemicals of S. terebinthifolius to mitigate hyperglycemia, oxidative stress, and inflammation in T2D.

Methodology
The study employed a multi-faceted approach involving phytochemical extraction, nanoparticle synthesis, in vitro cellular assays, and in vivo animal models.

• Synthesis and Characterization: Pink pepper (S. terebinthifolius) fruit extract was prepared via ultrasound-assisted extraction. Cerium oxide nanoparticles (CeriaNPs) were synthesized using a sol-gel method with ammonium hydroxide, followed by conjugation with the plant extract to form ST-CeriaNPs. The resulting nanoparticles were characterized using Dynamic Light Scattering (DLS) for size and zeta potential, Transmission Electron Microscopy (TEM) for morphology, Fourier-Transform Infrared (FTIR) spectroscopy for functional group interactions, X-ray Diffraction (XRD) for crystallinity, and X-ray Photoelectron Spectroscopy (XPS) for surface composition.
• Phytochemical Analysis: The extract was analyzed for total phenolic and flavonoid content using spectrophotometry and High-Performance Liquid Chromatography (HPLC-MS/MS) to identify specific compounds, including gallic acid, catechin, and masazino-flavanone.
• In Vitro Studies:
  • Cytotoxicity and Functionality: RINm5f β-cells were exposed to ST-CeriaNPs to assess viability (MTT assay) and glucose-stimulated insulin secretion.
  • Immunomodulation: RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS) to evaluate the modulation of pro-inflammatory (IL-1β, IL-6, TNF-α) and anti-inflammatory (IL-4, IL-10) cytokines.
• In Vivo Studies: Experimental T2D was induced in male C57BL/6J mice via streptozotocin (STZ) administration. Diabetic mice were divided into six groups: Normal Control, Diabetic Control, Diabetic + ST Extract, Diabetic + Ceria NP, Diabetic + ST-Ceria NP, and Diabetic + Metformin. Treatments were administered orally for four weeks.
• Physiological and Biochemical Assessments: Efficacy was evaluated through body weight monitoring, fasting blood glucose, Oral Glucose Tolerance Tests (OGTT), insulin levels, HbA1c, lipid profiles (TC, HDL, LDL, TG), liver enzymes (AST, ALT), and oxidative stress markers (SOD, CAT, GSH, MDA) in serum, pancreas, and liver tissues. Histopathological examination of pancreatic tissue was also conducted.

Key Contributions and Results
The study successfully developed and characterized ST-CeriaNPs, demonstrating a synergistic therapeutic effect superior to individual components or standard Metformin therapy in several metrics.

• Nanoparticle Properties: The ST-CeriaNPs exhibited a uniform spherical morphology with an average size of 199 ± 1.76 nm and a negative zeta potential (–32.6 mV), while the unmodified CeriaNPs exhibited a zeta potential of –32.2 mV. FTIR analysis confirmed the interaction between flavonoid functional groups in the extract and the nanoparticle surface, stabilizing the formulation.
• In Vitro Efficacy:
  • Insulin Secretion: ST-CeriaNPs significantly restored insulin secretion in STZ-stressed RINm5f cells. Under glucose stimulation, insulin release was enhanced in a dose-dependent manner, with significant improvements observed at concentrations >20 µg/mL.
  • Anti-inflammatory Action: In LPS-stimulated macrophages, ST-CeriaNPs significantly suppressed pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) by approximately 50–60% while upregulating anti-inflammatory cytokines (IL-4, IL-10).
• In Vivo Efficacy:
  • Glycemic Control: ST-CeriaNP treatment achieved a 60% inhibition rate of hyperglycemia levels after four weeks, outperforming ST extract (53%), Ceria NP (49%), and Metformin (32%). It also significantly lowered HbA1c and HOMA-IR indices.
  • Lipid and Liver Profile: The combination therapy effectively normalized lipid profiles, reducing Total Cholesterol, LDL-C, and Triglycerides while increasing HDL-C. It also significantly reduced elevated liver enzymes (AST, ALT), indicating hepatoprotection.
  • Oxidative Stress and Histology: ST-CeriaNPs restored antioxidant defenses (SOD, CAT, GSH) and reduced lipid peroxidation (MDA) in serum, pancreas, and liver tissues more effectively than other treatments. Histopathological analysis revealed that ST-CeriaNPs mitigated STZ-induced pancreatic islet necrosis and promoted the recovery of β-cell architecture.
  • Body Weight: Treated mice regained body weight, with the ST-CeriaNP group showing a 1.41-fold increase, contrasting with the 38.5% weight loss in untreated diabetic controls.

Significance and Claims
The authors claim that this work represents the first exploration of combining cerium oxide nanoparticles with Schinus terebinthifolius extract for diabetes treatment. The study highlights that the ST-CeriaNP formulation delivers synergistic benefits, achieving outcomes comparable to or exceeding Metformin in managing T2D symptoms. The proposed mechanism involves the dual action of CeO₂ nanoparticles, which scavenge free radicals via redox cycling, and the plant extract, which provides antioxidant phenolics and anti-inflammatory modulation.

The paper concludes that ST-CeriaNP is a promising nanotherapeutic strategy that addresses the multifactorial nature of T2D by simultaneously reducing hyperglycemia, oxidative stress, and inflammation while restoring insulin sensitivity and pancreatic integrity. However, the authors maintain a modest stance regarding clinical translation, noting that further investigation into molecular pathways, metabolite profiling, and long-term safety is required before translational and clinical applications can be pursued.