Rutin rut

Reagents: Protocatechuic (PRO), caffeic (CAF), p-coumaric (COU) and ferulic (FER) acids were purchased from Merck (Kenilworth, NJ, USA), chlorogenic (CHL) acid was obtained from Alfa Aesar (Haverhill, MA, USA), rosmarinic (ROS) acid, rutin (RUT) and quercetin (QUE) were acquired from Sigma Aldrich, luteolin (LUT) and kaempferol (KAE) were purchased from Roth (Dautphetal, Germany), while isoquercitrin (ISO) was obtained from HWI Analytik (Rülzheim, Germany). All HPLC gradient grade solvents (water, acetonitrile) were purchased from Merck. The calibration curve for this method with standard chromatogram, and retention times can be found in Supplementary Materials, Section S2.4 (Tables S4 and S5, Figure S51).

The flavonoids evaluated in the study were: ampelopsin (AMP, #42866), isoquercitrin (ISO, #17793) and rutin (RUT, #R5143), all obtained from Sigma-Aldrich (St. Louis, MO, USA). Flavonoid stock solutions were prepared in dimethyl sulfoxide—DMSO at 30 mg/mL. Calcium hydroxide PA (CH, Biodynamics, Ibiporã—PR/BR) at 1 mg/mL and chlorhexidine gluconate (CHX, Manipullis Pharmacy, Araçatuba—SP/BR) at 20 mg/mL were diluted in sterile deionized water as positive controls. All compounds were filtered in a 0.22 µm membrane filter and store at—20 °C. Table S1 shows the flavonoids and controls used in the study, their codes, synonyms, chemical structure, and molecular weight. All the following experiments described were performed in triplicate in three independent experiments (n = 9/group) [29 (link),30 (link)].

Five experimental water-ethanol solutions were prepared using a standard concentration (6.5%) of each of the following flavonoids: hesperidin (HES); quercetin (QUE); naringin (NAR); rutin (RUT) (Sigma Aldrich, St. Louis, MI, USA); grape seed (extraction from Vitis vinifera, high in proanthocyanidins) (PRO) (purity 95%) (Active Pharmaceutica, Palhoça, SC, Brazil). The purity, solubility index, hydrophobicity index, and the critical micelle concentration (CMC) of each flavonoid were kept in consideration for the formulation of the experimental pretreatment solutions used in this study (Table 1).
A specific composition for each flavonoid was obtained to allow the maximum availability of the active principle, but without jeopardizing their overall properties (Table 2) [22 (link)]. A placebo solution (PLA) containing only the vehicle used in the solution was obtained to quantify the effects of the active principles. A control group (CON) with the adhesive applied using none of the previous pretreatment solutions was also included in the study.