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.

The aim of this study was to investigate the potential of Amaranthus cruentus flavonoids (quercetin, kaempferol, catechin, hesperetin, naringenin, hesperidin, and naringin), cinnamic acid derivatives (p-coumaric acid, ferulic acid, and caffeic acid), and benzoic acids (vanillic acid and 4-hydroxybenzoic acid) as antioxidants, antidiabetic, and antihypertensive agents. An analytical method for simultaneous quantification of flavonoids, cinnamic acid derivatives, and benzoic acids for metabolomic analysis of leaves and inflorescences from A. cruentus was developed with HPLC-UV-DAD. Evaluation of linearity, limit of detection, limit of quantitation, precision, and recovery was used to validate the analytical method developed. Maximum total flavonoids contents (5.2 mg/g of lyophilized material) and cinnamic acid derivatives contents (0.6 mg/g of lyophilized material) were found in leaves. Using UV-Vis spectrophotometry, the maximum total betacyanin contents (74.4 mg/g of lyophilized material) and betaxanthin contents (31 mg/g of lyophilized material) were found in inflorescences. The leaf extract showed the highest activity in removing DPPH radicals. In vitro antidiabetic activity of extracts was performed with pancreatic α-glucosidase and intestinal α-amylase, and compared to acarbose. Both extracts exhibited a reduction in enzyme activity from 57 to 74%. Furthermore, the in vivo tests on normoglycemic murine models showed improved glucose homeostasis after sucrose load, which was significantly different from the control. In vitro antihypertensive activity of extracts was performed with angiotensin-converting enzyme and contrasted to captopril; both extracts exhibited a reduction of enzyme activity from 53 to 58%. The leaf extract induced a 45% relaxation in an ex vivo aorta model. In the molecular docking analysis, isoamaranthin and isogomphrenin-I showed predictive binding affinity for α-glucosidases (human maltase-glucoamylase and human sucrase-isomaltase), while catechin displayed binding affinity for human angiotensin-converting enzyme. The data from this study highlights the potential of A. cruentus as a functional food.

We developed a naringenin–hesperidin molar mixture (MIX–160) with proven antihyperglycemic and vasorelaxant activity in preclinical studies. A solid dosage form was manufactured to improve the bioavailability properties. In the current study, we sought to evaluate the oral preclinical toxicity of the MIX–160 dosage form, which showed no mortality or significant changes in the body weight, food consumption and tissue/organ mass in rats. Three daily oral doses (50, 300 and 2000 mg/kg of MIX–160) were assayed for 28 days. The results showed no structural abnormalities in the histological analysis and no significant changes (p > 0.05) in the liver biochemical markers (total bilirubin, AST and ALT) compared to the control group. The above findings showed that the MIX–160 dosage form did not exhibit relevant toxic effects, which suggests its potential safety as a drug candidate for clinical studies.