Inhibition of plant essential oils and their interaction in binary combinations against tyrosinase

Background Essential oils (EOs), derived from aromatic plants, exhibit properties beneficial to health, such as anti-inflammatory, anti-oxidative, antidiabetic, and antiaging effects. However, the effect of EOs and their interaction in binary combinations against tyrosinase is not yet known. Objective To evaluate the underlying mechanisms of EOs and their interaction in binary combinations against tyrosinas. Design We explored to investigate the inhibitory effect of 65 EOs and the interaction among cinnamon, bay, and magnolia officinalis in their binary combinations against tyrosinase. In addition, the main constituents of cinnamon, bay, and magnolia officinalis were analyzed by gas chromatography–mass spectrometry (GC–MS). Results The results showed that the most potent EOs against tyrosinase were cinnamon, bay, and magnolia officinalis with IC50 values of 25.7, 30.8, and 61.9 μg/mL, respectively. Moreover, the inhibitory mechanism and kinetics studies revealed that cinnamon and bay were reversible and competitive-type inhibitors, and magnolia officinalis was a reversible and mixed-type inhibitor. In addition, these results, assessed in mixtures of three binary combinations, indicated that the combination of cinnamon with bay at different dose and at dose ratio had a strong antagonistic effect against tyrosinase. Magnolia officinalis combined with cinnamon or bay experienced both antagonistic and synergistic effect in anti-tyrosinase activity. Conclusion It is revealed that natural EOs would be promising to be effective anti-tyrosinase agents, and binary combinations of cinnamon, bay, and magnolia officinalis might not have synergistic effects on tyrosinase under certain condition.

T yrosinase, a copper-containing enzyme, was reported to play a key role in the synthesis of melanins, which were the pigments responsible for skin color in human beings and enzymatic browning in nature (1). In human being, moderate melanin, catalyzed by tyrosinase, was synthesized to prevent skin lesions caused by ultraviolet radiation. However, the upregulation of tyrosinase activity and excessive accumulation of melanin would cause skin disorders and related diseases, such as freckles, malignant melanoma, and Parkinson's disease (2)(3)(4). In nature, fruits and vegetables browning, related with the increase of tyrosinase activity, was confirmed to shorten the shelf life of fruits and vegetables, which causes an unattractive appearance and unpredicted loss in nutritional Popular scientific summary • 65 plant essential oils have different inhibition against tyrosinas. Cinnamon, bay and magnolia offinalis have most potent inhibition than other EOs. Moreover, cinnamon and bay were reversible and competitive-type inhibitors, and magnolia officinalis was a reversible and mixed-type inhibitor. • Binary combinations of cinnamon, bay, and magnolia officinalis might not have synergistic effects on tyrosinase under certain condition. quality (5)(6)(7). Recent research indicated that kojic acid, widely used for skin whitening and hyperpigmentation preventing, would lead to skin irritation and mutagenic effect on human skin (8)(9)(10). Thus, searching for safe tyrosinase inhibitors of nature origin has attracted increasing attention in cosmetic and medicinal industries (11). Over the years, tyrosinase inhibitors of nature origin were considered free of harmful side effects (7,12). Therefore, using active constituents derived from natural plants is a promising strategy to improve tyrosinase inhibition activity. Essential oils (EOs), extracted from various aromatic plants, were secondary metabolites (13). They comprised different bioactive components, exhibiting anti-inflammatory, anti-oxidative, antidiabetic, and antiaging properties (14). Recently, the literature on tyrosinase inhibitors from natural source is extensive (12,15,16). It was indicated that Litsea cubeba EO showed inhibition against tyrosinase with an IC 50 value of 166.7 μg/mL (17). In addition, EO that extracted from the peel of C. sinensis oranges had the low response with 11.18 ± 3.34% inhibition at a concentration of 800 μg/mL (18). However, due to their inadequate potency, most of EOs have not yet used for anti-tyrosinase applications.
Aiming to develop more potent tyrosinase inhibitors from natural source, 65 commercial EOs that extracted from plants were evaluated for their anti-tyrosinase activity as single constituents, as well as in binary combinations in this study. Furthermore, the main components of potent EOs were analyzed by gas chromatography-mass spectrometry (GC-MS). The kinetics and the anti-tyrosinase mechanism of potent EOs were discussed.

Materials
Sixty-five commercial EOs, pure without additive, were purchased from Jingjing Biotechnology Co. (Guangzhou, China). The information of these EOs was listed in Table 1. Tyrosinase (EC 1.14.18.1) and kojic acid were purchased from Sigma-Aldrich (St. Louis, MO). Tyrosinase was dissolved with 0.05 M phosphate buffer solution (PBS, pH 6.81 ± 0.01) and then diluted to 1,500 U/mL. The final concentration of tyrosinase and L-Dopa in PBS was 37.5 U/mL and 1.0 mM, respectively. In addition, kojic acid was used as a standard compound. Other solvents and reagents that had analytical grade were purchased from Tansoole (Shanghai, China).

Tyrosinase inhibition assay
All the EOs and kojic acid were dissolved in dimethyl sulfoxide (DMSO) to obtain varying concentrations as required. Inhibitory effect of EOs at different concentrations against tyrosinase was determined as previously described (19). In brief, the mixture containing 85 μL PBS, 10 μL tyrosinase, and 5 μL EOs (DMSO in the blank) was first added to the 96-well plates at 0°C and preincubated for 10 min at 37°C. Second, 100 μL of L-Dopa was rapidly added in the 96-well plates to initiate the enzyme reaction monitored for 60s shake in the microplate reader (Multiskan GO, Thermo Scientific, USA). Finally, the change in absorbance at 475 nm during the 60s shake was tested and recorded. These assays were, respectively, conducted as triplicate, and the concentration required for 50% inhibition of each EO was determined as IC 50 value. The inhibition rates of EOs were tested at more than 5 concentrations. The IC 50 values were calculated by the Origin 9.0 software. Inhibition of EOs the tyrosinase reaction was calculated as follows: where A means the absorbance of samples (60s), B means the absorbance of samples (0s), C means the absorbance of the blank (60s), and D means the absorbance of the blank (0s).

Kinetic analysis of tyrosinase inhibition
As the method reported previously (19), the inhibition mechanism of bay, cinnamon, and magnolia officinalis was determined. Typically, a series of diluted inhibitor solutions were first prepared, at a constant L-Dopa concentration (2 mM). Second, the inhibition of bay, cinnamon, and magnolia officinalis against tyrosinase was measured with different concentrations of tyrosinase (the final: 0.0, 12.5, 25.0, and 37.5 U/mL), respectively. In addition, the inhibition kinetics of tyrosinase was tested by Lineweaver-Burk plots. A series of diluted inhibitor solutions were obtained, at a constant tyrosinase concentration (37.5 U/mL). The inhibition rates were determined with different concentrations of L-Dopa (the final: 1.5, 1.2, 0.9, 0.6, and 0.3 mM) by using the method reported previously.

Analysis of 65 EOs with GC-MS
Essential oils were analyzed with GC (TRACE 1300E, Thermo Scientific Corporation, USA) coupled to MS (ISQ Qd, Thermo Scientific Corporation, USA) using a TG-5 MS silica column (30 m × 0.25 mm; film thickness 0.25 μm). The procedure for analyzing GC-MS was shown as follows: helium was first used as the carrier gas, flowing with a rate of 1.0 mL/min. The oven temperature was second programmed at 60°C, with an increase of 5°C/min to 160°C (isotherm at 2 min), and then 10°C/ min to 260°C and held for 20 min. The mass spectra were recorded at 70 eV with a scanning range from 35 to 450 m/z. Composition (%) of EOs was calculated in software by using the peak normalization method. Comparing to Kovats retention indices and relative to a C 8 -C 40 n-alkanes standard, the peak identification of different constituents in EOs was determined. Identification of the EOs constituents was performed by comparing the acquired mass spectrum with NIST mass spectral library.

Statistical analysis
Initially, 65 EOs were tested individually. The three EOs that had the lowest IC 50 (cinnamon, bay, and magnolia officinalis) were mixed in five different proportions forming each binary combinations (9:1, 3:1, 1:1, 1:3, and 1:9). And then, three binary combinations were tested. In addition, five concentrations (2.8, 8.3, 25.0, 75.0, and 225.0 μg/mL) of three binary combinations were, respectively, determined in order to investigate the mode of interaction among cinnamon, bay, and magnolia officinalis in their binary combinations against tyrosinase. CompuSyn Version 2.0, generating the combination index-fraction affected (CI-Fa) curve, isobologram figure and median effect, was used to classify the anti-tyrosinase activity of cinnamon, bay, and magnolia officinalis in their binary combinations (20). The effects of cinnamon, bay, and magnolia officinalis in their binary combinations were classified according to CI as synergy (0.1 < CI < 0.9), additive (0.9 < CI < 1.1), and antagonism (1.1 < CI < 10) (21).

Inhibitory effect of 65 EOs against tyrosinase
It has been reported that several EOs and their main constituents were potential tyrosinase inhibitors (22).
However, systematic research of plant EO as natural tyrosinase inhibitors was still lacking. Therefore, we investigated the inhibitory effect of 65 EOs on tyrosinase in vitro. As described in Table 1 1). Taken together, cinnamon, bay, and magnolia officinalis were selected for further investigation of the anti-tyrosinase mechanism of action.

Mechanism study
To further investigate the inhibitory mechanism of cinnamon, bay, and magnolia officinalis on tyrosinase, the inhibition constants and inhibition types of cinnamon, bay, and magnolia officinalis on tyrosinase activity were determined by using L-Dopa as the substate. As shown in Fig. 1, the four lines were obtained from four different concentrations of cinnamon, bay, and magnolia officinalis. The lines were crossed on the origin. In addition, the slope of the line was gradually decreased with increasing EOs (cinnamon, bay, and magnolia officinalis) concentrations. These results indicated that the inhibition of cinnamon, bay, and magnolia officinalis on tyrosinase activity was reversible. As seen in Fig. 2, the inhibition kinetics of cinnamon, bay, and magnolia officinalis were analyzed by Lineweaver-Burk plots. The four lines were obtained from four different concentrations of cinnamon and bay ( Fig. 2A and B). The lines were crossed on a 1/V axis, indicating that cinnamon and bay exhibited competitive-type inhibition on tyrosinase, respectively. The results of these experiments showed that cinnamon and bay only bound free enzyme and not the enzyme-substrate complex. In addition, the four lines with different slopes were obtained from four different concentrations of magnolia officinalis (Fig. 2C). These lines were crossed on the third quadrant, suggesting that magnolia officinalis showed mixed-type inhibition.
To investigate whether cinnamon, bay, and magnolia officinalis inhibited tyrosinase activity by competitively forming enzyme-inhibitor (EI) complex or interrupting enzyme-substrate-inhibitor (ESI) complex in noncompetitive manner, we determined EI dissociation constants K i of cinnamon, bay, and magnolia officinalis and the ESI dissociation constants K is of magnolia officinalis. Inhibition type, and K i and K is values of cinnamon, bay, and magnolia officinalis were shown in Table 2. K i values of cinnamon, bay, and magnolia officinalis were 67.5, 55.1, and 667.5 μg/mL, respectively. Furthermore, a lower value of K is in comparison with K i demonstrated that there was weaker binding between enzyme and magnolia officinalis, which suggested preferred noncompetitive over competitive manner. It was confirmed that the cinnamon, bay, and magnolia officinalis had significantly tyrosinase inhibitory activities, indicating that they might be practically used as tyrosinase inhibitors.

Main constituents of cinnamon, bay, and magnolia officinalis.
In an attempt to comprehensively characterize the main constituents of the potential EOs that contribute to the anti-tyrosinase activity, three EOs (cinnamon, bay, and magnolia officinalis) were analyzed by GC-MS. As listed in

The interaction between cinnamon, bay, and magnolia officinalis in their binary combinations against tyrosinase
The three oils (cinnamon, bay, and magnolia officinalis) with best anti-tyrosinase activity were mixed in five different ratios to perform three binary compositions. The results were shown in Tables 4, 5, and 6. As seen from data in Table 4, the combination cinnamon + bay had a high combination index (CI >1.1), indicating that cinnamon combined bay at different dose (2.8, 8.3, 25, 75, and 225 μg/mL) and dose ratios (9:1, 3:1, 1:1, 1:3, and 1:9) had an antagonistic effect on tyrosinase. Furthermore, the result in Table 4 revealed that low dose of cinnamon + magnolia officinalis (2.8 and 8.3 μg/mL) showed an antagonistic effect on tyrosinase. However, moderate dose (25 μg/mL) and high dose (75 and 225 μg/mL) of cinnamon + magnolia officinalis experienced antagonistic, additive, and synergistic effects on tyrosinase. In addition, CI values of bay + magnolia officinalis at low dose (2.8 and 8.3 μg/mL) and different dose ratios against tyrosinase were ranged from 1.0 to 1.2, suggesting that the combination of bay + magnolia officinalis had antagonistic and additive effect on tyrosinase, respectively. Combination of bay and magnolia officinalis at 25 and 225 μg/mL showed additive and synergistic effect on tyrosinase. Bay combined with magnolia officinalis at 75 μg/mL and different dose ratios had a synergistic effect against tyrosinase. Linear retention index is obtained from https://webbook.nist.gov/chemistry/.

Discussion
Tyrosinase, known as monophenol and polyphenol oxidase, is responsible for synthesis of melanins in plants and animals (23). In the process of melanin biosynthesis, tyrosinase catalyzed the oxidation of mono-or di-phenolic compounds to corresponding L-Dopa and dopaquinones (24). Previous studies confirmed that several compounds, including arbutin, azelaic acid, and kojic acid, had been widely used as hyperpigmentation preventing agents and whitening agents (25). Nevertheless, kojic acid, widely used in the cosmetic products, had profound drawbacks, such as skin irritation and mutagenic effect on mammalian cells (26). Therefore, more attention is urgently needed to searching for safe tyrosinase inhibitors. In the present study, we investigated whether the EOs derived from natural plants as single constituent as well as in binary combinations had inhibitory effect on tyrosinase. Moreover, the kinetics and the anti-tyrosinase mechanism of potent EOs (cinnamon, bay, and magnolia officinalis) were determined by using L-Dopa as the substrate. Additionally, the main components of cinnamon, bay, and magnolia officinalis were analyzed by GC-MS. It was indicated that EOs and their main constituents had a wild range of bioactivities, such as antibacterial, antifungal, and anti-inflammatory activities (5,13). Herein, we investigated inhibitory effect of 65 EOs on tyrosinase in vitro. The results demonstrated that most of EOs showed different inhibitory activity against tyrosinase. Furthermore, pomelo, notopterygium, aloe, ginger, schizonepeta tenuifolia, and burdock showed higher inhibition than that of other EO from the families of Rutaceae, Apiaceae, Liliaceae, Zingiberaceae, Labiatae, and Compositae, respectively. Among these EOs, the most potent EOs against tyrosinase were cinnamon, bay, and magnolia officinalis with IC 50 values of 25.7, 30.8, and 61.9 μg/mL, respectively. Furthermore, GC-MS analysis showed that cis-cinnamaldehyde (90.85%), trans-cinnamaldehyde (86.03%), androsta-1,4,6-triene-3,17-dione (32.75%), and magnolol (31.28%) were the main constituents of cinnamon, bay, and magnolia officinalis, respectively. Previous studies confirmed that these compounds had anti-tyrosinase activity (27,28). Thus, cinnamon, bay, and magnolia officinalis might be responsible for anti-tyrosinase activity of these compounds.
As shown in Fig. 1, the inhibition of cinnamon, bay, and magnolia officinalis on tyrosinase activity was reversible. Moreover, further studies demonstrated that cinnamon and bay exhibited competitive-type inhibition on tyrosinase, and magnolia officinalis showed mixed-type  inhibition. Cinnamon and bay only bound free enzyme and not the enzyme-substrate complex. The inhibitory mechanism revealed that K i values of cinnamon, bay, and magnolia officinalis were 67.5, 55.1, and 667.5 μg/mL, respectively. It was confirmed that there was weaker binding between enzyme and magnolia officinalis, which suggested preferred noncompetitive over competitive manner. Taken together, these results demonstrated that the cinnamon, bay, and magnolia officinalis showed effectively anti-tyrosinase activity, indicating that they could be used as natural tyrosinase inhibitors. Our studies revealed that the combination cinnamon + bay had a high (CI >1.1), indicating that cinnamon combined bay at different dose (2.8, 8.3, 25, 75, and 225 μg/ mL) and dose ratios (9:1, 3:1, 1:1, 1:3, and 1:9) had an antagonistic effect on tyrosinase. Low dose of cinnamon + magnolia officinalis (2.8 and 8.3 μg/mL) at five dose ratios showed an antagonistic effect on tyrosinase. In addition, bay combined with magnolia officinalis at 75 and 225 μg/ mL and different dose ratios had synergistic and additive effects on tyrosinase. The possible reason for these was cis-and trans-cinnamaldehyde, and principal compounds of cinnamon and bay EO, mixed with different dose and different dose ratios exerted an antagonistic effect on tyrosinase. Combination of cis-cinnamaldehyde from cinnamon and major constituents of magnolia officinalis (androsta-1,4,6-triene-3,17-dione and magnolol) at 2.8 and 8.3 μg/mL exhibited an antagonistic effect on tyrosinase, respectively. Moreover, combination of trans-cinnamaldehyde from cinnamon with major constituents of magnolia officinalis at 75 and 225 μg/mL exhibited an antagonistic effect on tyrosinase, which showed synergistic and additive effect on tyrosinase. Herein, investigating the anti-tyrosinase effect of the combination of main constituents in bay, cinnamon, and magnolia officinalis EO may provide theoretical basis for searching novel natural tyrosinase inhibitors as whitening agents in cosmetics products. However, the interactions of combination of these compounds were currently investigated in the ongoing studies in our lab.
In summary, we indicated that natural tyrosinase inhibitors could be discovered from EOs of naturally edible plants. Among the EOs, cinnamon, bay, and magnolia officinalis significantly inhibited tyrosinase activity. The in vitro experiments demonstrated that cinnamon and bay were reversible and competitive-type inhibitors, and magnolia officinalis was a reversible and mixed-type inhibitor. Additionally, the further studies revealed that binary combinations of cinnamon, bay, and magnolia officinalis might not have synergistic effects on tyrosinase under certain condition. Collectively, these results demonstrated that cinnamon, bay, and magnolia officinalis EOs, promising tyrosinase inhibitors, could be used as whitening agents in cosmetics products.