3.4.3. Anti-Tyrosinase Assay

Assays of tyrosinase inhibition of the two plants EOs, as well as the 1:1 mixture, were carried out via measuring of L-DOPA chrome formation according to the described protocol of Batubara, et al. [61]. Briefly, the two EOs and a mixture of them (1:1, *w*/*w*), separately, were dissolved in a solvent with three certain concentrations (10, 100, and 250 μg/mL). The assays were performed by insertion of the following components: (a) phosphate buffer (120 μL, 20 mM, pH 6.8), (b) 20 μL sample, and (c) 20 μL mushroom tyrosinase (500 U/mL in 20 mM phosphate buffer) in 96-well plates. After 15 min of incubation at 25 ◦C, the intiation of reaction was occurred by insertion of 20 μL L-tyrosine solution (0.85 mM) for every well and followed by incubation for 10 min at room temperature. The activity of the enzyme was monitored at 475 nm using a Microplate reader (TECAN, Inc.). EGCG was used as a positive control. The calculation of the tyrosinase inhibition % was performed via the following equation:

$$\text{Tyrosinase inhibition (\%)}=[\text{(A}-\text{B)}-(\text{C}-\text{D})]/(\text{A}-\text{B})\times 100\tag{1}$$

where A is the absorbance of the control with the enzyme, B is the absorbance of the control without the enzyme, C is the absorbance of the test sample with the enzyme, and D is the absorbance of the test sample without the enzyme.

3.4.4. Anti-Hyaluronidase Assay

The fluorimetric Morgan–Elson assay method was performed according to Reissig, et al. [62] that modified by Takahashi, et al. [63]. In a brief description, a 5 μL of tested EOs and a mixture of EOs of the two plants (1:1, *w*/*w*), separately, were incubated for 10 min at 37 ◦C with bovine hyaluronidase (1.50 U) in 100 μL of 20 mM sodium phosphate buffer solution (pH 7.0), sodium chloride (77 mM), in addition to 0.01% bovine serum albumin (BSA). The assay reaction was initiated via adding the hyaluronic acid sodium salt (100 μL) from rooster comb (0.03% in 300 mM sodium phosphate, pH 5.35) to the incubation mixture, then the mixture was incubated at 37 ◦C for 45 min. The precipitation of undigested hyaluronic acid was carried out by 1 mL acidic solution of albumin, involving 0.1% BSA in sodium acetate (24 mM) and acetic acid (79 mM, pH 3.75). The mixture was stoped by allowing it for 10 min at room temperature, and fluorescence was detected using a Tecan Infinite microplate reader at 545 nm excitation and 612 nm emission EGCG was used as a positive control.

The percentage of the collagenase, elastase, and hyaluronidase inhibition was calculated via the following equation:

$$\text{Enzyme inhibition (\%)} = \left[1 - (\text{S/C}) \times 100\right] \tag{2}$$

where *S*: the corrected absorbance of the samples containing elastase inhibitor (the enzyme activity in the presence of the samples); and *C*: the corrected absorbance of controls (the enzyme activity in the absence of the samples).

The IC50, the concentration required to inhibit 50% of the enzyme under the assay conditions, was estimated from graphic plots of the dose-response curve for each concentration using Graphpad Prism software (San Diego, CA, USA).
