Diosmin

Rutin (quercetin 3-O-rutinoside) (2) was purchased from Alchimica s.r.o. (Czech Republic). Hesperidin (hesperetin 7-O-rutinoside) (3), narcissin (isorhamnetin 3-O-rutinoside) (4), diosmin (diosmetin 7-O-rutinoside) (5), and p-nitrophenyl β-d-glucoside (7) were purchased from Sigma-Aldrich. Isoquercitrin (quercetin 3-O-β-d-glucopyranoside) (1) and p-nitrophenyl rutinoside (8) were prepared as previously described (Weignerová et al. 2012 (link); Šimčíková et al. 2015 (link)).

Low molecular weight chitosan sample (average molecular weight <300 kDa; a degree of deacetylation = 83%) was produced from shrimp shells (Litopenaeus vannamei) using a UAE. Analytical reagents and standards used were: Folin–Ciocalteu reagent (2N), sodium carbonate (Na₂CO₃), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), potassium persulfate (K₂S₂O₈), ρ-iodonitrotetrazolium, Mueller-Hinton broth, sodium tripolyphosphate (TPP), gallic acid, chlorogenic acid, caffeic acid, eriocitrin, ellagic acid, ρ-coumaric acid, sinapic acid, diosmin, hesperidin, catechin and quercetin, HPLC grade methanol, acetic acid, and acetonitrile were purchased from Sigma-Aldrich (Toluca, State of Mexico, Mexico). Ultra-pure water was prepared in a Milli-Q water filtration system (Millipore, Bedford, MA, USA).

Diosmin (DM; MW: 608.54 g/mol, purity: ≥90%), nanohydroxyapatite (nHAp, <200 nm particle size), 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (EDC), fluorescein diacetate (FDA) and 3-(4,5-Dimethylthiazol-2-yl) 2,5-Diphenyltetrazolium Bromide (MTT) were obtained from Sigma Aldrich (St. Louis, MO, USA). C3H10T1/2 cells (mouse mesenchymal stem cells; mMSCs) were obtained from National Centre for Cell Science (NCCS), Pune, India. Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Lonza-BioWhittaker (Walkersville, MD, USA) and Fetal Bovine Serum (FBS) was purchased from Invitrogen^M (Waltham, MA, USA). 5-bromo-4-chloro-3′-indolyl-phosphate-nitro-blue tetrazolium (BCIP-NBT) was procured from Amresco® (OH, USA). Gelatin (Gel) and the antibodies for western blot analysis were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Westar Supernova-chemiluminescent substrate was purchased from Cyanagen (Bologna, Italy). Phosphate buffered saline (PBS) was procured from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). Acetic acid, ethanol, and other reagents used were of analytical grade.

Methylglyoxal (40% in water), glyoxal (40% in water), 2,2-diphenyl-1-picrylhydrazyl,2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), methanol (HPLC grade), acetonitrile (HPLC gradient grade and LC-MS grade), water (LC-MS grade), diosmin, diosmetin, hesperidin, hesperetin, Trolox, metformin hydrochloride, bovine serum albumin, DMSO, 98–100% formic acid, 2-methylquinoxaline, quinoxaline, o-phenylenediamine, 2, 4, 6-tripyridyl-s-triazine, iron(III) chloride hexahydrate, and iron(II) sulfate heptahydrate were purchased from Merck-Sigma-Aldrich (Sigma-Aldrich Sp. z o.o., Poznań, Poland); NaCl, KCl, Na₂HPO₄, and KH₂PO₄ (reagent grade) were obtained from Chempur (Piekary Śląskie, Poland); quercetin and rutin were from Extrasynthese (Genay Cedex, France); calcium dobesilate was purchased from PPF Hasco-Lek S. A. (Wroclaw, Poland). Water used in the study was glass-distilled and deionized. The stock solutions of standards (3 mM) were prepared by dissolving the appropriate amount of a reference compound in 5 mL of methanol or DMSO. Working standard solutions for the derivatization experiment in the range of 5–210 μM were made by mixing with 50% aq. (aqueous) methanol (v/v), filtered through hydrophilic Millex Syringe Filters (Durapore 0.22 μm; Millipore, Burlington, MA, USA) and stored at −20 °C.

HPLC-grade methanol (MeOH), HPLC-grade acetonitrile (ACN), dimethyl sulfoxide (DMSO), sodium carbonate (Na₂CO₃ > 99%), aluminium chloride (AlCl₃ > 99%), D-(+)-glucose, DL-lactic acid (90%), choline chloride (98%), and standard compounds: ethyl-o-vanillin - ISTD (99%), diosmin (95%), rutin (99%), chrysin (99%), morin hydrate (95%), myricetin ( > 96%), apigenin (97%), (–)-epicatechin (99%), (+)-catechin, vanillic acid (97%), syringic acid (97%), trans-p-coumaric acid (98%), o-coumaric acid (98%), ferulic acid (98%), and rosmarinic acid (97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chlorogenic acid (99%), trans-cinnamic acid (98%), quercetin hydrate (99%) and flavone (99%) were supplied by Acros Organics (Belgium). Protocatehuic acid (99%), caffeic acid (99%) and kaempferol as well as Folin–Ciocalteu phenol reagent (2N), D-(–)-fructose and sodium acetate (CH₃COONa) were supplied by Merck (Germany). Gallic acid (99%) was purchased from Carlo Erba (Italy). Ultrapure water (resistance above 18 MΩ cm) used was obtained from a Milli-Q water purification system.

Acetonitrile (ACN) and 2-propanol (IPA), HPLC grade, were purchased by Panreac —AppliChem (Darmstadt, Germany). Methanol (MeOH), HPLC grade, was acquired by Carl Roth (Carlsruhe, Germany). Hexane reagent grade 99% and acetic acid 99% were purchased by Sigma-Aldrich (Steinheim, Germany). Ultrapure water was provided by a Milli-Q purification system (Millipore, Bedford, MA, USA).
Sinapic acid 95%, gallic acid 98%, ferulic acid 98%, rosmarinic acid 98%, catechin 98%, epicatechin 97%, p-coumaric 98%, quercetin 98%, diosmin 97%, kaempferol 97%, vanillic acid 97%, and caffeic acid 98% were purchased from Sigma-Aldrich (Steinheim, Germany). Luteolin 98% was acquired from Santa Cruz Biotechnologies. Apigenin 97% was purchased from Alfa Aesar (Karlsruhe, Germany). α-Tocopherol 96%, β-tocopherol 96%, γ-tocopherol 96%, and δ-tocopherol 96% were purchased by Sigma-Aldrich (Steinheim, Germany). Stock standard solutions of each analyte (1000 mg/L) were solubilized in methanol and stored at −20 °C in dark brown glass bottles.

Polyphenolic standards (purity > 98%), including luteolin, apigenin, diosmin, apigenin-8-C-glucoside, naringenin, quercetin-3-O-glucoside, quercetin, kaempferol, myricetin, naringin, kaempferol-3-O-glucoside, luteolin-7-O-glucoside, p-coumaric acid, chlorogenic acid, and 4-hydroxybenzoic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The reference standards were accurately weighed and dissolved in methanol to prepare stock solutions (1 mg mL⁻¹). For the construction of calibration curves, a multi-compound working solution was prepared by appropriate dilution of stock solutions with methanol–water (80:20 v/v) to make concentrations in the range 0.02–5 µg·mL⁻¹. For the antioxidant tests, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,3,5-triphenyltetrazolium chloride (TPTZ), anhydrous ferric chloride, hydrochloric acid, potassium persulfate (K₂S₂O₈), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and sodium acetate were purchased from Sigma-Aldrich (MO, United States). Methanol (MeOH), ethanol, and water (LC-MS grade) were acquired from Carlo Erba reagents (Milan, Italy), whereas formic acid (98–100%) was purchased from Fluka (Milan, Italy).