Theanine

The concentration of amino acids was determined by the ninhydrin assay method [31 (link)]. A total of 2 mL of the above tea extraction solution was transferred to a 50 mL volumetric flask with 1 mL of reagent (20 g/L of ninhydrin and 0.8 g/L of SnCl₂·2H₂O) and 1 mL of buffer (0.067 M Na₂HPO₄ and 0.067 M KH₂PO₄, pH 8.0) and reacted for 15 min in a boiling water bath. The control flask contained 2 mL of distilled water, 1 mL of reagent, and 1 mL of buffer. The reacted solution was then transferred to a quartz cell with a black aperture (1 cm light-path) and colorimetric measurement was made on an HP8453E UV-VIS spectrophotometer (Hewlett Packard Company, Palo Alto City, CA, USA) at a wavelength of 570 nm. Theanine (Sigma-Aldrich, St. Louis, MO, USA) was used as the amino acid reference to make the calibration graph, and the amino acid concentration of the tea samples was expressed in terms of Theanine equivalent based on its absorbance at 570 nm on the calibration graph (Figure 1).

Catechin (C, ≥98%), galloCatechin (GC, ≥98%), galloCatechin-3-gallate (GCG, ≥98%), epiCatechin (EC, ≥98%), epiCatechin-3-gallate (ECG, ≥98%), epigalloCatechin (EGC, ≥98%), epigalloCatechin-3-gallate (EGCG, ≥98%), caffeine (CAF, ≥98%), theaflavins (TF, ≥95%), theaflavins-3-gallate (TF3G, ≥98%), theaflavin-3′-gallate (TF3′G, ≥98%), theaflavine-3,3′-digallate (TFDG, ≥98%), quercetin-3-O-galactoside (≥98%), myricetin-3-O-galactoside (≥98%), kaempferol-3-O-rutinoside (≥98%), vitexin (≥98%), astragaloside (≥98%), quercetin (≥98%), and kaempferol (≥98%) were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). l-alanine, l-arginine, l-aspartic acid, l-cystine, l-glutamic acid, glycine, l-histidine, l-isoleucine, l-leucine, l-lysine, l-methionine, l-phenylalanine, l-proline, l-serine, l-threonine, l-tyrosine, l-valine, l-asparagine, l-glutamine, theanine, γ-aminobutyric acid, and l-tryptophan were purchased from Sigma (Sigma-Aldrich Co., St. Louis, MO, USA). Anthrone reagent and ninhydrin/formic acid reducing agent were purchased from BeiJing DingGuochangSheng Biotech. Co., Ltd. (Bejing, China). Ethyl caprate (≥99%) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China), and n-alkane mixed standard C7-C40 was purchased from o2si (o2si smart solutions—an LGC Standards Company, Charleston, SC, USA).