Each 25-µg total protein of mucus adjusted with PBS to 70 µl was incubated with 10 µl of mushroom tyrosinase (Sigma-Aldrich) in potassium phosphate buffer pH 6.5 (5 U/reaction), and then 20 µl of 1 mg/ml L-DOPA (Sigma-Aldrich) in buffer was added. The reactions were monitored the absorbance at A475. Distilled water was used as a negative control and kojic acid as positive control. The inhibition was calculated from the formula: Inhibition (%) = [1 − (A475 in sample/A475 in control)] × 100%.

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