Catechin standard

Total flavonoid content was measured using the aluminum chloride assay as described by Marinova et al. [54 ] with some modifications. An aliquot (500 µL) of extracts or catechin standard (Sigma–Aldrich, St. Louis, MO, USA) solution (10, 20, 30, 40, 50, and 100 mg L⁻¹) was added to a 5 mL Eppendorf tube, containing 2 mL distilled water. To the diluted sample, 150 µL of 5% sodium nitrite (AppliChem, Darmstadt, Germany) was added. After 5 min, 150 µL of 10% aluminum chloride (Carl-Roth, Carlsruhe, Germany) was added. At the sixth min, 1 mL of 1 M sodium hydroxide was added, and the total volume was diluted to 5 mL using distilled water. The absorbance was measured against reagent blank at 510 nm, and total flavonoids were expressed as mg CE g⁻¹ DW (mg catechin equivalent/g dry weight) and calculated by the equation: T = CV/M, where T is the total flavonoid content, C is the concentration of catechin estimated in mg mL⁻¹, V is the volume of extract solution in ml, and M is the weight of extract in g.

Fresh tea leaves (superior clone-clone of PGL 15) were picked after the ages of 5 years, and 25 days after, the shoot tip was grown. Fresh tea leaves (superior clone-clone of PGL 15) with shoot tips and two leaves underneath (P + 2) were obtained from Universitas Gadjah Mada tea plantation, namely, Pagilaran Plantation site, Batang, Central Java, Indonesia (1000 above sea level), which plucked in September 2020. Tyrosinase (7,164 U/mg solid) was purchased from Sigma-Aldrich. β-Glucosidase (293 U/mg solid) was purchased from Xi'an Geekee Biotech (China). Catechin standard was purchased from Sigma-Aldrich. Methanol and acetonitrile used were HPLC grade and or analytical grade quality. Broken Orange Pekoe (BOP) black tea produced by Pagilaran Plantation used a commercial black tea to be compared with the Tyrosinase treatments.

The Total flavonoid content (TFC) of anchote flour extracts was estimated using the colorimetric method adopted from Tanvir et al. (2017 ). Briefly, 1 ml of extract with concentration of 0.1 g/ml was mixed with 0.3 ml of 5% sodium nitrite. After 5 min, 0.3 ml of 10% aluminum chloride was added, with 2 ml of 1 M sodium hydroxide after 6 min of incubation and with the immediate addition of 2.4 ml of distilled water to produce a total volume of 10 ml. The color intensity of the flavonoid–aluminum complex was measured at 510 nm using a spectrophotometer (V‐630; JASCO, Japan). Finally, the TFC was determined against (+)‐catechin standard ( Sigma‐Aldrich) (R²=0.988) as (+)‐catechin equivalent (CE) in a concentration range of 1.00–100.00 μg/ml, and the result was expressed as milligram of CE/g of anchote flour.