Chlorogenic acid

Deionized water was produced using Millipore Simplicty UV station (Merck Millipore, Burlington, MA, USA). Acetonitrile, formic acid, ethanol was purchased from VWR (Radnor, PA, USA). Chlorogenic acid, hyperoside, rutin, gallic acid were purchased from Carl Roth (Karlsruhe, Germany). L-arginine, Tween-80 and aluminum chloride were purchased from Sigma-Aldrich (Sant Louis, MI, USA). Querectin was bought from Borschagovsky CPP (Kyiv, Ukraine) and fructose—from LLC Company ”Ukrhimsyre” (Kharkiv, Ukraine). Blood glucose, high-density lipoprotein cholesterol (Ch-HDL) and low-density lipoprotein cholesterol (Ch-LDL) (Felitis-Diagnostics, Ukraine) insulin (DRG, Germany) and triacylglycerols (TAG, Lachema, Czech Republic) were determined in blood serum using standard sets of reagents. Chemical standards used for HPLC analysis were previously isolated and identified in the Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw.

Chlorogenic acid and narcissin (isorhamnetin-3-O-rutinoside), as analytical standards, were purchased from Roth GmbH (Karlsruhe, Germany) and Sigma-Aldrich Co. (St Louis, MO, USA), respectively. HPLC grade water, HPLC grade acetonitrile and acetate buffer were provided by the JT Baker–Avantor Performance Materials B.V. (Deventer, The Netherlands). Solvents used for determination of total flavonoid content were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland). Chitosan (CS) 80/500 and 80/1000, with a deacetylation degree of 80%, were supplied by HMC⁺ GmbH (Halle, Germany). (Hydroxypropyl)methyl cellulose (HPMC viscosity 2.600–5.600 cP) and all other chemicals were from Sigma–Aldrich Chemical Co.

Methanol and ethanol were from VWR (Darmstadt, Germany) and were like all other solvents used of HPLC grade. Trolox, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), Folin-Ciocalteu reagent, quercetin, (−)-epicatechin, procyanidin B1, procyanidin B2, procyanidin C1, p-coumaric acid, trans-cinnamic acid and phloridzin dihydrate were from Sigma–Aldrich (Taufkirchen, Germany). Gallic acid, (+)-catechin, chlorogenic acid, ferulic acid, coffeic acid, rutin trihydrate and acetic acid were purchased from Carl Roth (Karlsruhe, Germany). Avicularin was used from Phytolab (Vestenbergsgreuth, Germany). Protocatechuic acid, hyperoside, isoquercitrin, quercitrin and procyanidin A2 were from Extrasynthese (Genay, France). Reynoutrin was purchased from Carbosynth Limited (Berkshire, UK). 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) was from Fisher Scientific (Nidderau, Germany). HPLC grade water (18 MΩ) was prepared using a MicroPure purification system (Thermo Electron LED GmbH, Niederelbert, Germany). Sodium hydroxide, hydrochloric acid (Merck, Darmstadt, Germany), meta-phosphoric acid (Sigma–Aldrich) and all other chemicals were of analytical grade.

References used for the HPLC-MS analysis were purchased from Sigma Aldrich (St. Louis, MO, USA): Chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin, quercetin, isoquercitrin, quercitrin, hyperoside, kaempferol, myricetol, and fisetin, Roth (Karlsruhe, Germany): Ferulic acid, sinapic acid, gentisic acid, gallic acid, patuletin, luteolin or from Dalton (Toronto, ON, Canada): cichoric acid, caftaric acid. HPLC grade solvents, analytical grade acids used for mobile phases and Folin-Ciocâlteu reagent were purchased from Merck (Darmstadt, Germany), together with sodium carbonate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and aluminium chloride used for antioxidant assays. ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) ≥98% purity, potassium peroxodisulfate (≥99% purity), DPPH, and Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; ≥97% purity) also used in antioxidant tests were purchased from Sigma Aldrich (Schnelldorf, Germany). Gallic acid monohydrate (99.5%) was purchased from Serva, (Heidelberg, Germany).

Disodium hydrogen phosphate dodecahydrate was purchased from Bernd Kraft GmbH (Duisburg, Germany). Sodium dihydrogen phosphate monohydrate was from AppliChem GmbH (Darmstadt, Germany) and catechol was from ThermoFisher GmbH (Kandel, Germany). Aceton was used from VWR International LLC (Fontenay-sous-Bois, France). The standards for the HPLC analysis (chlorogenic acid, catechin, epicatechin, phloretin-2-glucoside, and quercetin 3-glucoside) and hydrochloric acid (25%) were from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Sodium carbonate was purchased from Grüssing GmbH (Filsum, Germany) and potassium peroxodisulphate was from Fisher Scientific UK Ltd. (Loughborough, UK). Folin-Ciocalteu’s phenol reagent, potassium dihydrogen phosphate, and nitric acid (65%) were purchased from Merck KgaA (Darmstadt, Germany). Galllic acid and 2,2’-azobis(2-methylpropionamidine) dihydrochloride were from Fisher Scientific GmbH (Schwerte, Germany). 2,2’-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, trolox, and fluorescein were purchased from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany). All of the chemicals were of analytical grade. Water was purified through a Milli-Q water system (PURELAB^®, Elga LabWater, Veolia Water Technologies GmbH, Celle, Germany) and was used for buffers, the extracting agents, and dilution of sample extracts.

To analyze the phenolic compounds, 20 mg of a lyophilized okra sample was extracted with 60% methanol (per analysis, Carl Roth GmbH, Karlsruhe, Germany) according to Neugart et al. [63 (link)]. For the quantitative analysis of the flavonoid glycosides and hydroxycinnamic acid derivatives, an HPLC series 1100 (Agilent Technologies, Waldbronn, Germany) was used, according to Neugart et al. [64 (link)]. The tentative identification of flavonoid glycosides and hydroxycinnamic acid derivatives was performed using an amazon SL ion trap mass spectrometer (Bruker Daltonics GmbH & Co. KG, Bremen, Germany) in negative ionization mode, according to Neugart et al. [64 (link)]. Standards (chlorogenic acid, quercertin 3-glucoside, and kaempferol 3-glucoside; Roth, Karlsruhe, Germany) were used for external calibration curves. Results are presented as mg g⁻¹ dry weight (DW).

Maslinic acid, corosolic acid, oleanolic acid, and ursolic acid were obtained from Phytolab (Vestenbergsgreuth, Germany), lithocholic acid (LCA), chenodeoxycholic acid (CDCA), oleic acid, and palmitic acid from Sigma Aldrich (St. Louis, MO, United States), stearic acid and 4-methoxycinnamic acid from Fluka (Buchs, Switzerland), chlorogenic acid from Carl Roth (Karlsruhe, Germany), and linoleic acid from Acros (Geel, Belgium). Eugenol was isolated from clove and the identity and purity tested by ATR-IR spectroscopy. All reference and standard compounds were obtained in a purity of ≥90%. They were dissolved to 1.0 mg/mL in MeOH. The triterpene acids (TTAs) used for the quantification were mixed and diluted to give a standard solution containing 100 μg/mL of each corosolic acid, Maslinic acid, oleanolic acid, and ursolic acid followed by a serial dilution down to 2 μg/mL.