Chrysin

Chrysin, a natural compound of cell culture grade was obtained from Sigma-Aldrich Company (St. Louis, MO, USA). Dimethylsulphoxide (Sigma-Aldrich, USA, 100%) was used for dissolving Chrysin and 5 mM stock concentration was prepared. Serial dilutions of drug were performed with the Dulbecco’s Modified Eagle's medium (DMEM) cell culture media to get variable concentrations used for in vitro studies. For subsequent experiments, the drug stock was stored in opaque container at 4 °C. The natural compound as well as the other reagents was of molecular biology grade.

MDA-MB-231 cells (5,000 cells/well) were seeded in 96-well plates and incubated for 24 h. Chrysin (Sigma-Aldrich, St Louis, MO, USA), NP or Chrysin-NPs were then added for a 48 h-incubation period. G-1 and G-15 (Cayman Chemical, Michigan, USA) were added for a 24 and 48 h-incubation period. MTT (Sigma-Aldrich) was added to the media for a further 3 h, and the supernatant was gently removed and discarded. DMSO (Sigma-Aldrich) was added and absorbance (560 nm) was determined using a microplate reader (Infinite M200 PRO; Tecan Inc., Grödig, Austria). The data were represented a mean ± standard deviation (SD, n = 4).

The animals in the vehicle group received 1 mL/kg/day of propylene glycol (Ajax Finechem Pty Ltd., Auckland, New Zealand) by oral gavage and 1 mL/kg/day of 0.9% normal saline solution by intraperitoneal (i.p.) injection. The animals in the D-gal group were given 50 mg/kg/day of D-gal (Sigma Aldrich, St. Louis, MO, USA) dissolved in 0.9% normal saline solution by i.p. injection. The animals in the chrysin 10 and 30 groups were administered with chrysin, which is 97% purity determined by HPLC analysis, and purchased from Sigma Aldrich, St. Louis, MO, USA, at 10 or 30 mg/kg/day dissolved in propylene glycol by oral gavage. The animals in the D-gal + chrysin 10 and D-gal + chrysin 30 groups received D-gal at a similar dose to the D-gal group and chrysin at equivocal doses as the chrysin 10 and 30 groups, respectively. All drugs were administered for 8 weeks. The animals were intraperitoneally injected with BrdU (100 mg/kg/day, Sigma Aldrich, St. Louis, MO, USA) dissolved in 0.9% normal saline solution at the first, second, and third day of drug administration.

Human cervical cancer (HeLa) cells were used as an in vitro cancer model during this study. HeLa cells were maintained in complete Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich; Merck, KGaA) containing 10% FBS (Sigma-Aldrich; Merck KGaA) and penicillin (100U/mL) (Sigma-Aldrich; Merck KGaA), and incubated at 37 °C with 5% CO₂.
Chrysin (powdered, mol wt. 254.241 g/mol) was procured from Sigma-Aldrich (Merck, KGaA), and stock solution was prepared with DMSO (78.67 mM) using DMSO (stock solution). Furthermore, sub-stock (1 mM) and concentrations (5, 10, and 15 µM) of Chrysin were prepared using the complete media as a diluent.

Silver nitrate (≥99%), sodium hydroxide (NaOH), bovine serum albumin (BSA), HPLC grade methanol, ethanol, formic acid, fetal bovine serum, Mueller–Hinton, Luria–Bertani, and Dulbecco’s Modified Eagle media, as well as L-asparagine (98%), L-arginine monohydrochloride (≥98%), L-glutamine solution (200 mM), sodium pyruvate solution (100 mM), and penicillin-streptomycin solution (1000 U/1 U per mL) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Pinocembrin (≥95%), pinobanksin-3-O-acetate (≥95%) (Pb-3-O-Ac), chrysin (≥95%), galangin (≥95%), and quercetin (≥95%) were purified from propolis collected in Ures, Sonora, Mexico (N 2927.1810, W 110 23.398) by chromatographic isolation procedures over silica gel 60 (0.015–0.040 mm; Merck KGaA), using progressive proportions of ethyl acetate in hexane as the mobile phase (Tedia Company, Fairfield, OH, USA). Caffeic acid phenethyl ester (CAPE) was synthesized according to the esterification of caffeic acid and phenethyl alcohol, based on the procedure described by Grunberger et al. [16 (link)].

Methanol (Chromosolv^TM for HPLC, ≥99.9%) and acetonitrile (UHPLC supergradient ACS quality) were obtained from PanReac AppliChem (Barcelona, Spain), formic acid (≥98%) from Sigma-Aldrich (St Louis, MO, USA), and hydrochloric acid (37%) from Fisher Chemical (Geel, Belgium). Water was purified by employing an Elix 3 system coupled to a Milli-Q instrument from Millipore Corporation (Bedford, MA, USA). The water was filtered with a 0.22 µm nylon membrane filter integrated into the Milli-Q instrument.
All the polyphenolic and phenolic acid compounds used in this work were of analytical grade and were obtained from Sigma-Aldrich, with the exception of hesperidin obtained from Glentham Life Sciences (Lorsham, United Kingdom), astilbin and caftaric acid from Biopurity Phytochemicals Ltd. (Chengdu, Sichuan, China), trans-coumaric acid and procyanidin C1 from Phytolab (Vestenbergsgreuth, Germany), diosmin, hesperetin, and catechol from AlfaAesar Chemicals (ThermoFisher, Kandel, Germany), naringin, naringenin, and epigallocatechin from Biosynth-Carbosynth (Berkshire, United Kingdom), procyanidins B2 and A2 from Extrasynthese (Genay, France), pinocembrin from Fisher Scientific (Madrid, Spain), tricetin and galangin from Cymit Quimica S.L. (Barcelona, Spain), and chrysin and pinobanksin from Merck (Darmstadt, Germany).

Ochratoxin
A (OTA), chrysin
(CHR), warfarin, iodipamide, hemin, bilirubin, human serum albumin
(HSA; product code: A1653), and rat serum albumin (RSA; product code:
A6272) were purchased from Merck (Darmstadt, Germany). Chyrsin-7-sulfate
(C7S) was synthesized as it has been previously reported.^{22 (link),27 (link)} All other reagents were analytical grade or HPLC grade.