Hydroxybenzoic acid

The samples (300 mg) were extracted with 3 ml of methanol/water (80/20, v/v), for 10 min by sonication at room temperature. After centrifugation at 8000 rpm for 5 min, the supernatant was removed, and the extraction was repeated two times in a similar way. The combined supernatants were evaporated to dryness by centrifugal evaporation. The residues were dissolved in 400 μl of methanol/water (80/20, v/v) and filtered through a 0.2 μm PTFE membrane filter. A 20 μl of the final solution was injected into the HPLC system.
Phenolic compounds were separated on a LUNA-PFP (2) (3 μm, 150 mm × 4.6 mm) column thermostated at 35°C. Mobile phase A consisted of methanol/water (10/90, v/v) containing 0.1% acetic acid, while methanol containing 0.1% acetic acid served as mobile phase B. The gradient elution was performed as follows: 0.0 min, 5% B; 6.5 min, 25% B; 30.5 min, 37% B; 35.0 min, 55% B; 37.0 min, 95% B; 44.0 min, 95% B; 45.0 min, 5% B and 50.0 min; 5% B for re-equilibration of the column. The flow rate was adjusted to 0.7 ml/min. The injection volume was 20 μl. Phenolic compounds were monitored at 280 and 320 nm. For quantification, standards (Sigma–Aldrich, United States) of two subgroups of phenolic acid, viz., hydroxybenzoic acid (protocatechuic acid and p-hydroxy-benzoic acid) and hydroxycinnamic acid (p-coumaric acid, ferulic acid, and sinapic acid) were employed.

The ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) diammonium salt, potassium peroxodisulfate, 2,4,6-tris(2-pyridyl)-s-triazine, Trolox^®, aminoguanidine hydrochloride, methylglyoxal solution, L-arginine, hexane, 5-hydroxymethyl-L-furfural, BHT (2,6-Di-tert-butyl-4-methylphenol), (+)-catechin, rutin, quercetin, D-glucose, citric, L-ascorbic, oxalic, gallic and hydroxybenzoic acid were purchased from Sigma-Aldrich (Switzerland). Acetone and trifluoroacetic acid were bought from Acros Organics (France). Methanol and acetonitrile were obtained from Fine Chemicals (Netherlands), ethyl acetate from Alfa Aesar (Germany). Acetic and sulfuric acids were from Thermo Scientific (Germany). Lycopene, β-carotene, lutein and zeaxanthin were bought from Extrasynthèse (France) and absolute ethanol from Alcosuisse (Switzerland). Fructose and saccharose were obtained from Merck (Germany).
Folin and Ciocaulteu reagent, sodium dihydrogen phosphate mono-hydrate, di-sodium hydrogen phosphate anhydrous and sodium azide were purchased from Chempur (Poland), BSA (Bovine Serum Albumin) from Pol-Aura (Poland). The pectinase Pectinex Ultra SP-L was obtained from Novozymes (Denmark).
Deionized water (Milli-Q purification system, BlancLabo, Switzerland) was used for chromatography.
All reagents used were of analytical grade or higher. All solvents were of HPLC grade.

Chitosan (MW 200 kDa, the degree of deacetylation 85%) was purchased from Qingdao Baicheng Biochemical Corp. (Qingdao, China). The materials, including 3-chloro-2-hydroxypropyltrimethyl ammonium chloride, sodium hydroxide, cumaric acid, ascorbic acid, ferulic acid, p-coumaric acid, caffeic acid, gallic acid, salicylic acid, hydrochloric acid, and hydroxybenzoic acid were purchased from Sigma-Aldrich Chemical Corp. (Shanghai, China). sodium hydroxide, isopropyl alcohol, and ethanol were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the chemical solvents and reagents were obtained from commercial sources and used as received.

AFB₁ and FB₁ standard solutions (purity > 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The phenolic compounds gallic acid, sinapic acid, chlorogenic acid, caffeic acid, syringic acid, vanillin, vanillic acid, hydroxybenzoic acid, hydroxylcinnamic acid, p-coumaric acid, benzoic acid, DL-3-phenyllactic acid, 1,2-dihydroxybenzene, 3,4-dihydroxyhydrocinnamic, and DL-p-hydroxyphenyllatic acid were provided from Sigma-Aldrich (St. Louis, MO, USA). Phenyllactic acid was obtained from BaChem (Weil am Rhein, Germany). Ferulic acid was purchased from MP Biomedicals (Santa Ana, CA, USA) and protocatechuic acid from HWI pharma services (Ruelzheim, Germany). Acetonitrile (ACN) (99%), methanol (99%), ethyl acetate (EA) (99%) and formic acid (FA) (99%) used for liquid chromatography were of HPLC-grade and obtained from VWR Chemicals (Radnor, PA, USA). Magnesium sulfate (MgSO₄), ammonium formate and sodium chloride (NaCl) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sepra^TM C18-E (50 μm, 65 Å) was purchased from Phenomenex (Madrid, Spain). Microbiological media such as potato dextrose broth (PDB), potato dextrose agar (PDA), Man Rogosa Sharpe (MRS) broth and MRS Agar were obtained from Liofilchem Bacteriology Products (Roseto degli Abruzzi, Italy). Deionized water (<18 MΩ cm resistivity) was obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA).

Both gibberellic acid and indole-3-acetic acid were purchased from MB Cell, Seoul, Korea. External standards were purchased from different sources: hydroxybenzoic acid from Extrasynthese (Genay, France) and benzoic acid, catechin, caffeic acid, chlorogenic acid, epicatechin, quercetin, and rutin from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA).

The antioxidant standards: quercetin, morin, rutin, pyrogallol, mandalic acid, hydroxy benzoic acid, phloroglucinol and chlorogenic acid were obtained from Sigma-Aldrich France. From the same firm galanthamine (from Lycoris Sp.) was also obtained. Follin-ciocalteu reagent, sodium carbonate, 5,5-dithio-bis-nitrobenzoic acid (DTNB) and 2,2-Diphenyle-1 picrylhydrazyle (DPPH) were purchased from Sigma Aldrich CHEMIE GmbH USA. Aluminum chloride, sodium nitrite, sodium hydroxide, ethanol, methanol, ascorbic acid, 2,2′-azino-bis-3 ethylbenzothiazoline-6-sulfonic acid (ABTS) were bought from sigma Aldrich Germany. Phosphate buffer (pH 8), butyryl thiocholine iodide, acetylthiocholine iodide, acetyl cholinesterase from Electric eel (type-VI-S) and butyryl cholinesterase from equine used in enzyme inhibition assays were obtained from Sigma-Aldrich USA. All the chemicals used were of analytical grade except the HPLC solvents which were HPLC grade and were purchased from DaeJung Korea. They were used as such without any further purification. Distilled water used for HPLC analysis and antioxidant assays were prepared in biochemistry lab using automatic water still (Daihen labtech, china). The HPLC solvents were subjected to sonication (Elmasonic, Model E 30 H) to remove air bubbles before use.

AChE (Electron eel type-VI-S) and Aquine BChE along with their substrates acetylthiocholine iodide and butyrylthiocholine iodide, and potassium phosphate buffer (pH 8.0) were purchased from Sigma Aldrich St. Louis, MO, USA. Antioxidant chemicals: DPPH, ABTS, ascorbic acid, malic acid, morin, epigallocatechin gallate, pyrogallol, rutin, quercetin, chlorogenic acid, mandelic acid, hydroxy benzoic acid, and gallic acid along with Folin–Ciocalteu regent were acquired from Sigma Aldrich, Darmstadt, Germany. Donepezil and scopolamine were also purchased from Sigma Aldrich (USA). All these chemicals used were of analytical grade; however, the HPLC solvents were of HPLC grade.