Potencial bioativo de nanopartículas de peju acetilado contendo óleo essencial de citrus sinensis: avaliações in vitro e in vivo

Abstract

The essential oil of Citrus sinensis is a terpenoid mixture mainly composed of d-limonene and exhibits several biological activities described in the literature, including anti-inflammatory, antioxidant, antibacterial, and antiarrhythmic properties. However, it is still underutilized as a therapeutic compound by the pharmaceutical industry due to its limitations, such as low water solubility, susceptibility to oxidative processes, and poor absorption in the gastrointestinal tract. An alternative to mitigate these issues is the encapsulation of C. sinensis essential oil in polymeric nanoparticles, as they have the potential to improve the bioavailability of the active ingredient, protect the molecule from degradation in the gastrointestinal tract, and allow controlled release, reducing its degradation and clearance in biological systems. Thus, the present study aimed to nanoencapsulate C. sinensis essential oil (EO) using acetylated polysaccharides extracted from cashew gum as the polymeric matrix (acCGP@EO) and to evaluate the biological potential of the produced nanosystem. The nanoparticles were produced by nanoprecipitation and evaluated regarding their encapsulation efficiency, hydrodynamic diameter, polydispersity index, zeta potential, and morphology using transmission electron microscopy. Considering the purpose of testing the acCGP@EO in animal models, we first evaluate the cellular toxicity through the MTT assay. Results evidenced that the EO and acCGP@EO did not show baseline cytotoxicity at the tested concentrations, evidencing the safety of the non-nanostructured EO and the produced acCGP@EO nanoparticles. Also, the acCGP@EO nanoparticles protected cells from oxidative stress induced by hydrogen peroxide. After demonstrating the non-cytotoxicity and the cytoprotective potential of the produced nanoparticles, it was evaluated if this nanosystem could be absorbed into the gastrointestinal system. Results evidenced the presence of voluminous multivesicular bodies in the enterocyte's periapical cytoplasm, suggesting an increase in the absorptive capacity of these cells. Furthermore, a significant enlargement of the apical intercellular space was observed, indicating a possible absorption of acGCP@EO nanoparticles via paracellular transport. The biological potential of the produced acCGP@EO nanoparticles was assessed by evaluating their effect in an animal model of hypertension. The antihypertensive activity was assessed using normotensive Wistar rats and Spontaneously Hypertensive Rats (SHR) treated for 30 days with free essential oil or acCGP@EO in a dosage of 75 mg/kg. During treatment, the systolic blood pressure was monitored weekly by tail-cuff plethysmography. At the end of the treatment, water and food intake, as well as urine and feces excretion, were evaluated. Biochemical and histopathological tests were conducted to evaluate the cardiometabolic effect of the treatment. To assess the nephroprotective potential of the treatments, plasmatic and urinary levels of creatinine, urea, uric acid, total proteins, sodium, and potassium were measured. Hepatic and renal tissues were also analyzed in terms of their histopathological and immunohistochemical characteristics. The results demonstrated that the acCGP@EO nanoparticles effectively encapsulated the essential oil from C.sinensis, achieving an encapsulation efficiency of 75.18% and a hydrodynamic diameter of 161 nm. The polydispersity index was found to be 0.141, indicating a monodisperse nanosystem. The zeta potential was -19.17 mV, indicating a negative superficial charge that is attributed to the intrinsic characteristics of the acCGP molecule. Results from systolic blood pressure evidenced that EO and acCGP@EO had significant anti-hypertensive effects. The analysis of the plasma and urinary metabolites evidenced that EO and acCGP@EO nanoparticles promoted natriuresis without altering the plasmic levels of sodium, suggesting improvement of renal function. Results also evidenced a recovery of the glomerular filtration rate in the SHR animals treated with accGP@EO, confirmed by increased creatinine and urea clearance. This result highlights the best performance of the nanosystem compared to the non-nanostructured EO. The treatment with acCGP@EO nanoparticles was also efficient in reducing the lipidic peroxidation in the kidney and liver tissues. Treatment with EO and acCGP@EO nanoparticles was also efficient in increasing superoxide dismutase and catalase activity in the kidney and liver tissues of the SHR animals. Immunohistochemistry findings indicated a more pronounced anti-inflammatory effect of acCGP@EO nanoparticles in reducing hepatic and renal TNFα levels in SHR animals, demonstrating the nephroprotective and hepatoprotective potential of the produced nanoparticles. Altogether, results demonstrate that the proposed nanoparticle production methodology was effective, and the nanoencapsulation improved the bioactive potential of essential oil from Citrus sinensis, promoting absorption in the gastrointestinal tract, as well as antihypertensive, anti-inflammatory, nephroprotective, and hepatoprotective effects