Quercetin dihydrate

The initial screening of compound library was conducted using two strains of C. violaceum as model bacterium, and two assays (violacein extraction and resazurin staining) in parallel as recently optimized in [26 (link)]. For the screening assays, overnight cultures of C. violaceum were diluted in LB broth supplemented with yeast extract (LBY) (0.1% w/v, Sigma Aldrich, St. Louis, MO, USA). Culture of C. violaceum CV026 was further supplemented with 0.5 µM N-(β-ketocaproyl)-l-Homoserine lactone (3-O-C₆-(l)-HSL, Cayman chemicals, Ann Arbor, MI, USA) to induce the violacein production. Bacteria (10⁶ CFU/mL) was exposed to compounds (400 µM) in a total volume of 200 µL in 96-microtiter well plates (Nunc, Roskilde, Denmark), and incubated at 27 °C, with shaking at 200 rpm for 24 h. DMSO was added in untreated control wells and bacteria-free wells with only LBY were included controls. Additionally, autoinducer-free wells containing only bacteria were included in C. violaceum CV026 plates. Plates were prepared in duplicates. Quercetin dihydrate (Carl Roth GmbH, Karlsruhe, Germany) at 400 µM was used as positive control in violacein extraction assay, and azithromycin (Cayman chemicals, Ann Arbor, MI, USA) at 10 µM in viability assay. Both control compounds were prepared in DMSO. DMSO concentration was 2.5% throughout the experiments.

Extrusion trials were performed using commercial chokeberry (Aronia melanocarpa) pomace powder (CPP) (Aronia Original Naturprodukte GmbH, Dresden, Germany) [20 (link)]. Chemicals and reagents of analytical purity grade were obtained from Merck KGaA (Darmstadt, Germany), unless stated otherwise. Amyloglucosidase (E-AMGDFPD, from Aspergillus niger, 36,000 U/g), α-amylase (E-PANAA, from pig pancreas 100,000 U/g), protease (E-BSPRPD from Bacillus licheniformis 9000 U/g), Celite 545 and endo-arabinanase (EC 3.2.1.99, from A. niger, 9 U/mg) were from Megazyme (Bray, Ireland) and Amberlite FPA53 and Ambersep 200 from Rohm and Haas Europe (Frankfurt, Germany). Thermostable α-amylase (Thermamyl 120 L, EC 3.2.1.1, from B. licheniformis, 120 KNU/g), protease (Alcalase 2.5 L, EC 3.4.21.62, from B. licheniformis, 2.5 AU/g), and Amyloglucosidase (AMG 300 L, EC 3.2.1.3, from A. niger, 300 AGU/g) were a gift from Novozymes, (Bagsværd, Denmark). Rotihydroquant C5 and D used for Karl Fischer titration as well as cyanidin-3-O-glucoside (≥96%), cyanidin chloride (≥97%), 5-caffeoylquinic acid (≥97%), quercetin-3-O-glucoside (≥99%), and quercetin dihydrate (≥99%) used as PP standards were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Ultrapure water was used for all experiments.

Acetonitrile, methanol, ortho-phosphoric acid (HPLC grade), rutin, hyperoside, hypericin and hyperforin were purchased from Sigma-Aldrich, Germany for phytochemical profiling. Quercetin dihydrate (≥98%) was obtained from Carl Roth (Karlsruhe, Germany). Hydroxyethyl cellulose (HEC), hydroxy propylmethyl cellulose (HPMC), sodium carboxymethyl cellulose (NaCMC), and Pluronic F-127 were purchased from Sigma-Aldrich, Germany for the formulations. Ketamine HCl 5% (Rotex medica, Trittau, Germany), and Panthenol® 2% cream (Nile Co. for Pharmaceuticals and Chemical Industries, Egypt) were obtained for the in vivo experiments. Formaline, xylene, hematoxylin and eosin stain, and Masson’s trichrome stain were sourced from Sigma-Aldrich, Germany for the histopathology. All other chemicals and reagents were of analytical grade.