Many studies describe different pharmacological effects of flavonoids on experimental animals and humans. Nevertheless, few ones are confirming the safety of these compounds for therapeutic purposes. This study aimed to investigate the preclinical safety of naringenin, naringin, hesperidin, and quercetin by in vivo, in vitro, and in silico approaches. For this, an MTT-based cytotoxicity assay in VERO and MDCK cell lines was performed. In addition, acute toxicity was evaluated on Wistar rats by OECD Guidelines for the Testing of Chemicals (Test No. 423: Acute Oral Toxicity-Class Method). Furthermore, we used the ACD/Tox Suite to predict toxicological parameters such as hERG channel blockade, CYP450 inhibition, and acute toxicity in animals. The results showed that quercetin was slightly more cytotoxic on cell lines (IC₅₀ of 219.44 ± 7.22 mM and 465.41 ± 7.44 mM, respectively) than the other citroflavonoids. All flavonoids exhibited an LD₅₀ value > 2000 mg/kg, which classifies them as low-risk substances as OECD guidelines established. Similarly, predicted LD⁵⁰ was LD₅₀ > 300 to 2000 mg/kg for all flavonoids as acute toxicity assay estimated. Data suggests that all these flavonoids did not show significant toxicological effects, and they were classified as low-risk, useful substances for drug development.

The purpose of this study was to develop, optimize, and fully validate a high-sensitivity methodology using UHPLC-MS/MS to simultaneously quantify hesperidin and naringenin in microsamples (100 µL) of murine plasma after intragastric administration of single pure flavonoids and a mixture. The optimization process allowed for high sensitivity with detection limits of approximately picogram order using an electrospray ionization (ESI) source in negative mode and an experiment based on multiple reaction monitoring (MRM). The validation parameters showed excellent linearity and detection limits, with a precision of less than 8% and a recovery of over 90%. This methodology was applied to compare the pharmacokinetic parameters for the administration of hesperidin and naringenin in individual form or in the form of a mixture. The results showed an absence of significant effects (p > 0.05) for Tmax and Cmax; however, the AUC presented significant differences (p < 0.05) for both flavonoids when administered as a mixture, showing an improved absorption ratio for both flavonoids.

Introduction: Propolis has been used traditionally for different human diseases and even recently as dental biomaterials because of its antibacterial, antimycotic, and anti-inflammatory properties. However, a proper correlation between in vitro and in vivo anti-inflammatory properties has not been clearly established. Methods: The composition of propolis was determined by high-performance liquid chromatography–ultraviolet mass spectrometry (HPLC-UV-MS). Viability of ethanolic propolis solution was evaluated by thiazolyl blue tetrazolium bromide (MTT) assay on murine macrophages. The anti-inflammatory properties were assessed both in vitro through the enzyme-linked immunosorbent assay (ELISA) quantification of various cytokines and in vivo by induced edemas. Results: Chemical analysis showed pinocembrin, pinobanksin-3-O-acetate, and pinobanksin-3-O-propionate as the main components of propolis. Macrophage viability was high (106%) when propolis was used up to 50 µg/mL. ELISA studies showed a reduction in the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) up to 145 pg/mL, 350 pg/mL, and 210 pg/mL, respectively, while the anti-inflammatory cytokines (IL-10 and IL-4) were increased up to 833 pg/mL and 446 pg/mL. Finally, edema was reduced on paw and ear mice by 9% and 22%, respectively. Conclusion: Mayan propolis has strong in vitro anti-inflammatory properties without compromising macrophage viability, resulting in a low-to-mild in vivo anti-inflammatory response.