3.8. Anti-Enzymatic Activities

Acetylcholinesterase (AChE, Type-VI-S, EC 3.1.1.7, 222 U/mg) inhibitory activity of extracts were determined according to previously described Ellman’s colorimetric method [59]. Acetylthiocholine iodide was employed as the substrate to assay the inhibition of AChE. The reaction mixture contained 130 μL of 100 mM sodium phosphate buffer (pH 8.0), 20 μL of test sample solution and 20 μL of AChE (0.36 U/mL), which were mixed and incubated for 15 min at 25 °C. The reaction was then initiated via the addition of 40 μL of the following mixture (freshly prepared): 20 μL 0.5 mM DTNB (5,5′-dithiobis (2-nitrobenzoic acid) and 20 μL acetylthiocholine iodide (0.71 mM). The hydrolysis of acetylthiocholine iodide was monitored by following the formation of yellow 5-thio-2-nitrobenzoate anion at 412 nm every 10 sec for 10 min using a 96-well microplate reader under a constant temperature of 25 °C, which resulted from the reaction of 5–50-thiobis-2-nitrobenzoic acid with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide. The percent inhibition of acetylcholinesterase enzyme was calculated using the equation:

where, Abs_control is the absorbance of the assay using the buffer instead of inhibitor (sample) and Abs_sample is the absorbance of the sample extracts. Neostigmine bromide was used as the positive control [49]. Phosphate buffer was used as the blank. Each standard or sample solution was analyzed in triplicate. The concentration of the extracts which caused 50% inhibition of the tyrosinase activity (IC50) was calculated by nonlinear regression analysis.

The elastase inhibition of seaweeds extracts was investigated in TRIS buffer solution following the method of Eun et al. [60], with some modifications [61]. Briefly, 100 µL of 0.1 M Tris-HCl buffer solution (pH 8.0), 25 µL of elastase (1 U/mL in TRIS buffer) and 20 µL sample extracts were incubated for 15 min at 25 °C, before adding the substrate to begin the reaction. After incubation time, 40 µL of 0.5 mM N-Succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN) solution in water was added. Following this, absorbance at 410 nm was monitored for 20 min using a microplate reader under a constant temperature of 25 °C. Finally, elastase inhibition was calculated in percentage using the equation:

where, Abs_control is the absorbance of the assay using the buffer instead of inhibitor (sample) and Abs_sample is the absorbance of the sample extracts. Quercetin was used as the positive control [61]. Tris-HCl buffer was used as the blank. Each standard or sample solution was analyzed in triplicate. The concentration of the extracts which caused 50% inhibition of the tyrosinase activity (IC50) was calculated by nonlinear regression analysis.

Tyrosinase inhibitory assay was performed according to the method previously described by [61], with some modifications using 3,4-Dihydroxy-L-phenylalanine (L-DOPA) as substrate. A volume of 20 µL of sample, 20 µL of mushroom tyrosinase solution (100 U/mL in phosphate buffer) and 80 µL of phosphate buffer (pH = 6.8) were mixed and pre-incubated at 37 °C for 5 min. Then, 90 µL of L-DOPA (2 mg/mL water) was added. The formation of dopachrome was immediately monitored for 20 min at 475 nm in a microplate reader under constant temperature of 37 °C. The percent inhibition of tyrosinase enzyme was calculated using the equation:

where, Abs_control is the absorbance of the assay using the buffer instead of the inhibitor (sample) and Abs_sample is the absorbance of the sample extracts. Kojic acid was used as the positive control. Phosphate buffer was used as the blank. Each standard or sample solution was analyzed in triplicate. The concentration of the extracts which caused 50% inhibition of the tyrosinase activity (IC50) was calculated by nonlinear regression analysis.