Gallic acid monohydrate

The concentrations of gallic acid, methyl gallate and gallotanin in GEGR were measured as previously described [19 ,20 ]. Gallic acid monohydrate (IUPAC name; 3,4,5-trihydroxybenzoic acid, MW: 170.12 g/mol, Sigma-Aldrich Co., St. Louis, MO, USA), methyl gallate (IUPAC name; methyl 3,4,5-trihydroxybenzoate, MW: 184.15 g/mol, Sigma-Aldrich Co.) and gallotanin (IUPAC name; 3,5-dihydroxy-2-(3,4,5-trihydroxybenzoyl)oxy-6-[(3,4,5-trihydroxybenzoyl)oxymethyl]oxan-4-yl] 3,4,5-trihydroxybenzoate, MW: 1701.20 g/mol, Sigma-Aldrich Co.) were used as standard compounds during analysis of the main components of GEGR. The wavelengths of the maximum absorption of pure gallic acid, pure methyl gallate, commercial gallotannin, and gallnut extract were 212/257, 214/268, 213/278 and 212/275 nm, respectively. The UV-VIS spectra of pure gallic acid, pure methyl gallate, pure gallotannin, and the gallnut extract showed two bands at 212–214 nm and 257–278 nm, which were both assigned to the π→π* transitions of the given aromatic units and C = O groups in the UV-VIS region. Finally, the UV-Vis spectra were analyzed using a curve-resolving technique based on linear least-squares analysis to fit the combination of the Lorentzian and Gaussian curve shapes.

The following analytical grade or chemically pure reagents were purchased from commercial sources for this study: 30% hydrogen peroxide (H₂O₂), sodium acetate trihydrate (CH₃CO₂Na·3H₂O), ferrous sulfate heptahydrate (FeSO₄·7H₂O), sodium nitrite (NaNO₂), disodium hydrogen phosphate dodecahydrate (Na₂HPO₄·12H₂O), sodium dihydrogen phosphate dihydrate (NaH₂PO₄·2H₂O), ferric chloride hexahydrate (FeCl₃·6H₂O), anhydrous sodium carbonate (Na₂CO₃), potassium acetate (CH₃CO₂K), aluminum chloride (AlCl₃). Potassium ferricyanide (K₃[Fe(CN)₆]), salicylic acid sodium, gallic acid monohydrate, Folin-ciocalteu reagent, 2,6-di-tert-butylphenol, Rutin (C₂₇H₃₀O₁₆), t-butyl terephthalate phenol, Tris buffer, 2,4,6-Tri(2-pyridyl)-1,3,5-triazine (TPTA) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma chemicals.

Analytical reagent-grade (AR-grade) chemicals were used for all experiments without purification. Silver nitrate (AgNO₃), iron (III) nitrate nonahydrate (Fe(NO₃)₃·9H₂O), ethanol (C₂H₅OH; 99.9%), tetraethyl orthosilicate (TEOS), sodium carbonate, gallic acid monohydrate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), potassium persulfate (K₂Cr₂O₇), and ammonium hydroxide (NH₄OH) were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Green tea bags (Jeju company, Korea) were purchased from the local market.

Total polyphenol contents were estimated following the Folin-Ciocalteu method of Baba and Malik [18 (link)] with some modifications. Ten microliter sample was mixed with 150 μL of distilled water in 96-well microplate and mixed thoroughly with 25 μL Folin-Ciocalteu reagent (Sigma-Aldrich Corp., St. Louis, MO, USA) for 3 min. Then, the mixture was added to 100 μL 20% (w/v) sodium carbonate (Ajax Finechem, Auckland, New Zealand) and allowed to incubate at room temperature for 60 min in the dark. The absorbance of blue-complex was measured at 650 nm using a microplate reader (Tecan Sunrise, Männedorf, Switzerland). Gallic acid monohydrate (125, 250, 500, 750, 1000 μL/mL; Sigma-Aldrich Corp.) and deionised water were used as standard and blank, respectively. The total polyphenol contents of samples were expressed as milligram of gallic acid equivalents (GAE) in 100 g sample.

Ultra-high performance liquid chromatography (UHPLC, 1290 Infinity Quaternary LC System, Agilent, Santa Clara, CA, USA) was used to separate and identify the phenolics and flavonoids. The chromatographic system conditions were set as follows: mobile phase, 0.03 M orthophosphoric acid (A) and methanol HPLC grade (B); detector, UV 360 nm; column, C18 column (5.0 μm, 4.6 mm inner diameter [ID] × 250 mm); column oven temperature, 35 °C; and flow rate, 1.0 mL/min. Gradient elution was performed as follows: 0–10 min, 10 % B; 10–15 min, 50 % B; 15–20 min, 100 % B; and finally 5 min for washing. Linear regression equations were calculated using Y = aX ± b, where X is the concentration of the related compound and Y the peak area of the compound obtained from UHPLC [19 (link)]. The linearity was established by the coefficient of determination (R²). All flavonoids (rutin hydrate ≥94.0 %; quercetin ≥95.0 %; kaempferol ≥97.0 %; (+) catechin ≥99.0 %; naringenin 98.0 %; apigenin ≥95.0 %) and phenolic acids (gallic acid monohydrate ≥99 %; ferulic acid ≥99 %; trans-Cinnamic acid ≥99 %; chlorogenic acid ≥95 % and caffeic acid >98 %) standards were purchased from Sigma-Aldrich, Malaysia.

Gallic acid monohydrate, quercetin, and 5,7-dimethoxyflavone (DMF) were purchased from Sigma Aldrich (St. Louis, MI, USA). All the media, including Mueller–Hinton broth, tryptic soy broth, and brain heart infusion broth, were purchased from Himedia (Mumbai, India). Folin–Ciocalteu reagent was purchased from LOBA Chemie (Mumbai India). All the solvents used in this study were analytical grade.

Folin–Ciocalteu reagent, gallic acid monohydrate, ethanol, ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt), potassium persulfate (K₂S₂O₈), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), aluminum chloride (AlCl₃), sodium nitrite (NaNO₂), sodium hydroxide solution (NaOH) and quercetin were supplied from Sigma-Aldrich (Milan, Italy). Anhydrous sodium carbonate (Na₂CO₃) was supplied from Carlo Erba (Milan, Italy). All reagents were of analytical grade.