Anti-Oxidant, Anti-Aging, and Anti-Melanogenic Properties of the Essential Oils from Two Varieties of Alpinia zerumbet

Here, we investigated the anti-oxidant and anti-aging effects of essential oils (EOs) from the leaves of Alpinia zerumbet (tairin and shima) in vitro and anti-melanogenic effects in B16F10 melanoma cells. The anti-oxidant activities were performed with 2,2-diphenyl-1-picrylhydrazyl (DPPH); 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS); nitric oxide; singlet oxygen; hydroxyl radical scavenging; and xanthine oxidase. The inhibitory activities against collagenase, elastase, hyaluronidase, and tyrosinase were employed for anti-aging. The anti-melanogenic was assessed in B16F10 melanoma cells by melanin synthesis and intracellular tyrosinase inhibitory activity. The volatile chemical composition of the essential oil was analyzed with gas chromatography-mass spectrometry (GC/MS). The EO was a complex mixture mainly consisting of monoterpenes and sesquiterpenes. The results revealed that tairin and shima EOs showed strong anti-oxidant activities against DPPH and nitric oxide, hydroxyl radical scavenging activity, and xanthine oxidase inhibition. Compared to shima EO; tairin EO exhibited strong anti-aging activity by inhibiting collagenase, tyrosinase, hyaluronidase, and elastase (IC50 = 11 ± 0.1; 25 ± 1.2; 83 ± 1.6; and 213 ± 2 μg/mL, respectively). Both EOs inhibited intracellular tyrosinase activity; thus, reducing melanin synthesis. These results suggest that tairin EO has better anti-oxidant/anti-aging activity than shima EO, but both are equally anti-melanogenic.


Introduction
Melanin, the major skin pigment, plays a crucial role in protecting human skin against ultraviolet (UV) radiation. However, overproduction and accumulation of melanin can result in pigmented patches and skin discolorations such as melasma, freckles, post-inflammatory melanoderma, and solar lentigo [1]. Melanin biosynthesis is regulated by melanogenesis enzymes, such as tyrosinase, which catalyzes two different reactions. In the initial step of melanin synthesis, L-tyrosine is hydroxylated to L-3,4-dihydroxyphenylalanine (L-DOPA, O-diphenol product). L-DOPA is oxidized further to the corresponding O-quinone. Thus, tyrosinase is an important regulator and the rate-limiting enzyme for melanogenesis [2]. Tyrosinase also regulates the browning of vegetables and foods. Therefore, melanin synthesis and tyrosinase inhibitors are used commonly in food processing and in cosmetics for melanin hyperpigmentation and skin whitening. Moreover, skin disorders are associated with increased dermal enzymatic activity, including collagenase, hyaluronidase, elastase, and tyrosinase [3][4][5][6]. Thus, inhibitors of these enzymes are increasingly important ingredients in cosmetics and medications to protect the skin against hyperpigmentation and skin aging.
UV radiation exposure induces formation of excess reactive oxygen species (ROS), which can interact with proteins, lipids, and DNA, and alter cellular functions, thus causing aging-related disorders or melanogenesis [7,8]. Recently, melanogenesis has been reported to produce hydrogen peroxide and other ROS in melanocytes [9]. This observation has increased interest in identifying natural antioxidants that inhibit skin aging and melanogenesis.
Alpinia zerumbet (Pers.) Burtt and Smith (alpinia), a member of the Zingiberaceae family, is a perennial plant that is distributed widely in subtropical and tropical regions and is used in folk medicine. . Tairin is a taller plant with long stems, whereas shima is shorter with a bushy structure. The leaves of both plants are used in the preparation of a traditional food, mu-chi, and are thought to prevent common cold [10].
We previously demonstrated that alpinia leaves have anti-oxidant and anti-microbial activities [11,12]. We also reported that alpinia can inhibit advanced glycation end products, HIV-1 integrase, and neuraminidase (NA) [13,14]. Essential oils (EO) from alpinia also have been reported to have various biological activities such as a myorelaxant and anti-spasmodic agent in the rat ileum [15], anti-nociceptive in mice [16], and anti-hypertensive and cardiovascular disease preventative in rats [17]. In addition, alpinia EOs are associated with anti-microbial and larvicidal activity [18,19].
In this study, we examined the anti-oxidant and anti-aging activity of EOs isolated from the leaves of two varieties of alpinia. Furthermore, we evaluated the inhibitory activity of these samples against melanin biosynthesis in B16F10 melanoma cells.

Hydroxyl and Nitric Oxide Radical Scavenging Activities
The hydroxyl radical scavenging activity of tairin and shima EOs was determined as the percent inhibition of hydroxyl radical formation in the Fenton reaction. The scavenging activity of tairin and shima EOs was compared with that of ascorbic acid. Tairin EO (IC50 = 69 ± 2.0 μg/mL) had significantly higher (p < 0.05) hydroxyl radical scavenging activity and was more potent than ascorbic acid (IC50 = 72 ± 1.2 μg/mL), whereas shima EO had an IC50 = 74 ± 1.2 μg/mL ( Table 2).
Nitric oxide, generated from sodium nitroprusside in aqueous solutions, interacts with oxygen to produce nitrite ions, which can be measured by the Griess reaction. The EOs from tairin and shima showed scavenging activity between 66 and 704 μg/mL. The percent inhibition increased with increasing concentrations of EO. Tairin EO inhibited nitric oxide formation better (IC50 = 66 ± 1.6 μg/mL) than shima EO (IC50 = 704 ± 1.5 μg/mL) ( Table 2). However, ascorbic acid inhibited nitric oxide better than both EOs, with an IC50 value of 14 ± 0.8 μg/mL.

Tyrosinase and Hyaluronidase Inhibition Assay
Tyrosinase is a multifunctional, glycosylated, copper-containing oxidase that catalyzes the first two steps of mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. In this study, we evaluated ability of EOs from tairin and shima to inhibit tyrosinase. Tairin EO (IC50 = 25 ± 1.2 μg/mL) inhibited tyrosinase better than shima EO (IC50 = 28 ± 1.5 μg/mL). However, kojic acid inhibited tyrosinase better than both EOs (IC50 = 10 ± 0.7 μg/mL) (Figure 2A).
The hyaluronidases are a group of enzymes distributed throughout the animal kingdom. In recent years, hyaluronidase has received attention owing to its ability to regulate hyaluronan metabolism. Hyaluronidase inhibitors are potent regulators that maintain hyaluronidase homeostasis and may serve as anti-inflammatory agents. Our results indicate that tairin EO is a stronger inhibitor (IC50 = 83 ± 1.6 μg/mL) of hyaluronidase than shima EO (IC50 = 150 ± 0.8 μg/mL) ( Figure 2B).

Figure 1. Tairin and shima essential oil (EO)-mediated collagenase (A) and elastase (B)
inhibition. Values shown represent the mean ± standard error from three independent experiments. One-way ANOVA was used for the comparison of multiple group means, followed by the Duncan test (p < 0.05). Letters with different superscripts indicate samples that are significantly different (p < 0.05) from each other. IC, inhibitory concentration.

Figure 2.
Tairin and shima essential oil (EO)-mediated tyrosinase (A) and hyaluronidase (B) inhibition. Values shown represent the mean ± standard error from three independent experiments. One-way ANOVA was used for the comparison of multiple group means, followed by the Duncan test (p < 0.05). Letters with different superscripts indicate samples that are significantly different (p < 0.05) from each other. IC, inhibitory concentration.

Xanthine Oxidase Inhibition Assay
Xanthine oxidase is a flavoprotein that catalyzes the oxidation of hypoxanthine to xanthine and generates superoxide and uric acid. Xanthine oxidase inhibitors may be useful for the treatment of skin aging and gout, which is caused by the generation of uric acid and the superoxide anion radical. Our results indicate that tairin EO significantly inhibits xanthine oxidase (IC50 = 70 ± 0.6 μg/mL) and is a stronger inhibitor than shima EO (IC50 = 86 ± 0.4 μg/mL) (Figure 3). Values shown represent the mean ± standard error from three independent experiments. One-way ANOVA was used for the comparison of multiple group means, followed by the Duncan test (p < 0.05). Letters with different superscripts indicate samples that are significant different (p < 0.05) from each other. IC, inhibitory concentration.

Cell Viability
To analyze the effects of tairin and shima EOs on cell viability, B16F10 melanoma cells were treated with varying concentrations of EO for 48 h. As shown in Figure 4, the tairin and shima EOs did not affect the cell viability up to 50 μg/mL; however, treatment with 100 μg/mL caused a 26% ± 3% and 30% ± 3% decrease in cell viability, respectively. Thus, the tairin and shima EOs were used at concentrations up to 50 μg/mL to evaluate their effects on melanin content and intracellular tyrosinase activity. shown represent the mean ± standard error for three independent experiments. One-way ANOVA was used for the comparison of multiple group means, followed by the Duncan test (p < 0.05). Letters with different superscripts indicate samples that are significantly different (p < 0.05) from each other.

Effects of Tairin and Shima EOs on Melanin Content
To determine the anti-melanogenic activity of tairin and shima EOs, their effect on melanin content was evaluated in B16F10 melanoma cells. The B16F10 melanoma cells were treated with varying concentrations of tairin and shima EOs and then stimulated with IBMX for 48 h. As shown in Figure 5, tairin and shima EOs dose-dependently inhibited melanin content. At 50 μg/mL, melanin synthesis was inhibited 55% ± 6% and 48% ± 7%, respectively. Kojic acid, the positive control, inhibited melanin content 54% ± 7%. These results demonstrated that both tairin and shima EOs decreased the melanin content without any effect on the cell viability. Values shown represent the mean ± standard error for three independent experiments. A one-way ANOVA was used for the comparison of multiple group means, followed by the Duncan test (p < 0.05). Letters with different superscripts indicate samples that are significant different (p < 0.05) from each other.

Inhibition of the Intracellular Tyrosinase Activity by Tairin and Shima EOs
To evaluate whether tairin and shima EOs inhibit the intracellular tyrosinase activity, the B16F10 melanoma cells were treated with varying concentrations of the EOs for 48 h, followed by incubation with L-DOPA. Our data indicate that, following treatment with 20 μg/mL tairin and shima EOs, tyrosinase activity was inhibited by 41% ± 1% and 31% ± 6%, respectively. At a concentration of 50 μg/mL, both tairin EO (72% ± 1%) and shima EO (71% ± 1%) inhibited tyrosinase activity more potently than that by the positive control kojic acid (49% ± 3%) ( Figure 6). These results indicate that tairin and shima EOs could be applicable in cosmetics as skin-whitening agents for the treatment of pigmentation disturbances. Values shown represent the mean ± standard error for three independent experiments. One-way ANOVA was used for the comparison of multiple group means, followed by the Duncan test (p < 0.05). Letters with different superscripts indicate samples that are significant different (p < 0.05) from each other.

Discussion
The safety of natural products used in drugs, health foods, and cosmetic ingredients is a major concern. Several studies have explored the use of extracts from the leaves of Aesculus hippocastanum and Bunge [20,21], the gel of Aloe vera [22], the oil of Camellia japonica L. [23], and the rhizomes of Zingiber officinale L. [24] to prevent skin abnormalities and hyperpigmentation. In this study, we first determined the free radical scavenging activities of tairin and shima EOs. Our study demonstrated that tairin had stronger anti-oxidant activity than shima, with the exception of singlet oxygen scavenging activity. The GC/MS analysis showed tairin and shima EOs contain the major components of monoterpenes and sesquiterpenes. Previous studies have been reported that monoterpenes, such as terpinen-4-ol, 1,8-cineol, and γ-terpinene, showed anti-oxidant activities [11,12,25,26]. The strong anti-oxidant activity from tairin and shima EOs may be attributed from these components. It is known that UV radiation induces free radical formation in the skin, which is linked directly to the onset of skin photodamage and biological damage. Thus, our results suggest that tairin and shima EOs may be useful anti-oxidant sources to prevent UV-induced damage.
Next, we highlighted the anti-aging activity of EOs from tairin and shima through collagenase, elastase, hyaluronidase, and tyrosinase enzyme inhibition assays, and compared these results with those of oleanolic acid, which is a skin-protective pentacyclic triterpene. UV light stimulates collagenase expression, which causes matrix protein degradation and, subsequently, skin photoaging [27]. Elastase and hyaluronidase are proteolytic enzymes in the dermis that are responsible for the degradation of elastin and hyaluronan, respectively, in the extracellular matrix. Loss of elastin is a major part of what cause visible signs of aging (wrinkles, sagging) in the skin. Additionally, in human melanocytes, melanin synthesis is regulated by tyrosinase, leading to skin diseases. Our results indicate that tairin EO was a stronger tyrosinase, elastase, and hyaluronidase inhibitor than shima EO.
To our knowledge, the inhibitory effect of tairin and shima EOs on melanogenesis is reported here for the first time. IBMX is a well-known stimulator of melanogenesis, leading to tyrosinase synthesis and increased melanin levels [28]. We found that the EOs decreased melanin content in melanoma cells more effectively than the commercial melanin synthesis inhibitor (kojic acid). In order to understand better the potential use of tairin and shima EOs to reduce melanogenesis, we also evaluated the inhibition of intracellular tyrosinase activity. The present study indicated that tairin and shima EOs strongly inhibited intracellular tyrosinase activity. Thus, these EOs may be good candidates for use in skin-whitening materials.
In addition, we previously found a few RAC/CDC42-activated kinase 1 (PAK1)-blocking compounds in extracts of alpinia [29] and recently have revealed that PAK1 is essential for melanogenesis as well as the aging process and oncogenesis [30,31]. Thus, it is most likely that the anti-aging and anti-melanogenic effects of alpinia EOs could be partly attributed to their PAK1-blocking activity.

Extraction of EO from Alpinia Leaf
The EOs from fresh tairin and shima leaves (500 g) were isolated by steam distillation for 4 h. The distillate was extracted with diethyl ether, and the solvent was removed carefully under vacuum at 35 °C. The obtained EOs were dissolved in methanol and dimethyl sulfoxide (DMSO) and kept under refrigeration until use.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The compounds of EO were identified using DB-5MS fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm; Agilent Technologies, J & W Scientific Products, Folsom, CA, USA). The carrier gas was helium and the GC oven temperature program was as follows: 80 °C hold for 1 min, increase 10 °C/min to 220 °C, followed by increase to 330 °C at 20 °C/min and hold for 6 min. The injector and detector temperatures were set at 250 °C and 280 °C, respectively, and the injection volume was 1.0 μL in the splitless mode. Mass spectra were scanned form m/z 50-600 amu and the electron impact ionization energy was 70 eV. Quantitative determinations of EO components were done based on the peak area measurements. Retention indices were determined relative to the retention times of a series of n-alkane standards (C7 to C31, Restek Corporation, PA, USA), and compared with published values [11,12].

DPPH Radical Scavenging Assay
The DPPH radical scavenging assay was performed according to Boskou et al. [32]. DPPH (40 μL) and sodium acetate buffer (80 μL, 0.1 M, pH 5.5) were added to samples (80 μL) containing a range of EO concentrations (10, 100, and 250 μg/mL). The mixture was incubated at room temperature in the dark for 30 min. The absorbance was measured at 517 nm using a microplate spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). BHT was used as positive control.

ABTS Radical Scavenging Assay
The ABTS radical scavenging assay was carried out using a method previously described by Re et al. [33]. The working solution was prepared by mixing 7 mM ABTS and 2 mM K2S2O8 in equal quantities and allowing them to react in the dark overnight (more than 16 h) at room temperature. The solution was diluted with water to obtain an absorbance of 0.70 ± 0.03 units at 734 nm. The working ABTS solution (200 μL) was mixed with 20 μL of the samples containing varying EO concentrations (10, 100, and 250 μg/mL), and the absorbance was measured at 734 nm after 6 min of incubation at room temperature. BHT was used as a positive control.

Hydroxyl Radical Scavenging ( • OH) Assay
The hydroxyl radical scavenging assay was performed as described by Ozyurek et al. [34]. The reaction mixture for the deoxyribose assay contained the following reagents in a final volume of 1 mL: 200 μL of KH2PO4-KOH (100 mM), 200 μL of deoxyribose (15 mM), 200 μL of FeCl3 (500 μM), 100 μL of EDTA (1 mM), 100 μL of ascorbic acid (1 mM), 100 μL of H2O2 (10 mM), and 100 μL of sample. The reaction mixtures were incubated at 37 °C for 1 h. At the end of the incubation period, 1 mL of 2.8% (w/v) trichloroacetic acid (TCA) was added to each mixture, followed by the addition of 1 mL of 1% (w/v) thiobarbituric acid (TBA). The solutions were heated in a water bath at 90 °C for 15 min until a pink color developed, and the absorbance of the resulting solution was measured at 532 nm. Ascorbic acid was used as a positive control.

Singlet Oxygen Scavenging ( 1 O2) Assay
The production of singlet oxygen was determined by monitoring p-nitrosodimethylaniline (RNO) bleaching using a spectrophotometric method previously reported by Chakraborty and Tripathy [35]. Singlet oxygen was generated by reacting NaOCl and H2O2, and the bleaching of RNO was measured. The reaction mixture contained 45 mM phosphate buffer (pH 7.1), 50 mM NaOCl, 50 mM H2O2, 50 mM histidine, 10 μM RNO, and the test sample in a final volume of 2 mL. The reaction was incubated at 30 °C for 40 min, and the decrease in RNO absorbance was measured at 440 nm. Rutin was used as a positive control.

Nitric Oxide Scavenging Assay
Nitric oxide radical scavenging activity was estimated using the Griess Illosvoy reaction [36]. The reaction mixture (300 μL), containing 10 mM SNP (200 μL), phosphate-buffered saline (50 μL), and the test sample (50 μL), was incubated at 25 °C for 150 min. After incubation, 50 μL of the reaction mixture was mixed with 100 μL of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for complete diazotization. NED (100 μL) was added, and the mixture was incubated at 25 °C for 30 min, until a pink chromophore could be observed in diffused light. The absorbances of these solutions were measured at 540 nm against the corresponding blank solutions. Ascorbic acid was used as the positive control, while methanol was used as the control for calculation.
The % scavenging capacities of DPPH, ABTS, hydroxyl radical, singlet oxygen, and nitric oxide were calculated using the formula: % scavenging capacity = [(Acontrol − Asample)/Acontrol] × 100 (1) where Acontrol is the absorbance of the control without the test sample, and Asample is the absorbance of the sample.
All experiments were carried out in triplicate. The % radical scavenging activity was plotted against the corresponding extract concentration to obtain the IC50 value.

Collagenase Inhibition Assay
The collagenase inhibition assay was performed by measuring FALGPA hydrolysis using a previously described method [37]. The assay was performed in 50 mM tricine buffer, containing 400 mM NaCl and 10 mM CaCl2 (pH 7.5). Clostridium histolyticum collagenase (ChC) and the synthetic substrate, N- acryloyl)-Leu-Gly-Pro-Ala (FALGPA) were dissolved in the tricine buffer for use at initial concentrations of 0.8 U/mL and 2 mM, respectively. The EOs was incubated with the enzyme in the buffer for 15 min before adding the substrate to start the reaction. The final reaction mixture (150 μL total volume) contained tricine buffer, 0.8 mM FALGPA, 0.1 U ChC, and 25 μg EO samples. Water and methanol were used as negative controls, while oleanolic acid was used as a positive control. After incubating with the substrate for 20 min, collagenase activity was measured at 340 nm.

Elastase Inhibition Assay
Elastase inhibition was assayed using the method of Kransoe et al. [38] with slight modifications. Inhibition was determined by measuring the intensity of the solution color following elastase-mediated SANA cleavage. Briefly, 1 mM SANA was prepared in 0.1 M Tris-HCl buffer (pH 8.0). The solution (200 μL) was added to a stock sample solution (20 μL). The solutions were vortexed and preincubated for 10 min at 25 °C, and then 20 μL of elastase from porcine pancreas (0.03 U/mL) was added. After vortexing, each solution was placed in a water bath at 25 °C for 10 min. The absorbance was measured at 410 nm. Negative controls were performed with water and methanol, while oleanolic acid was used as a positive control.

Hyaluronidase Inhibition Assay
Inhibition of hyaluronidase was determined as previously described [39]. A 5 μL test sample was pre-incubated with bovine hyaluronidase (1.50 U) in 100 μL of solution containing 20 mM (pH 7.0) sodium phosphate buffer, 77 mM sodium chloride, and 0.01% bovine serum albumin (BSA) for 10 min at 37 °C. The assay was initiated by adding 100 μL of hyaluronic acid sodium salt from rooster comb (0.03% in 300 mM sodium phosphate, pH 5.35) to the incubation mixture, followed by incubation for 45 min at 37 °C. Undigested hyaluronic acid was precipitated with acid albumin solution (1 mL), composed of 0.1% BSA in 24 mM sodium acetate and 79 mM acetic acid (pH 3.75). The solution was allowed to stand at room temperature for 10 min, and the absorbance was measured at 600 nm. Oleanolic acid was used as the positive control.
The percent collagenase, elastase, and hyaluronidase inhibition were calculated by: Enzyme inhibition activity (%) = (1 − B/A) × 100 (2) where A is the enzyme activity without sample, and B is the activity in the presence of the sample.

Tyrosinase Inhibition Assay
Tyrosinase inhibition was determined by measuring DOPA chrome formation as previously described [40]. In brief, EO samples were dissolved in solvent to varying concentrations (10, 100, and 250 μg/mL). Samples were prepared in a 96-well plate, and the components were added as follows: phosphate buffer (120 μL, 20 mM, pH 6.8), 20 μL sample, and 20 μL mushroom tyrosinase (500 U/mL in 20 mM phosphate buffer). After incubation at 25 °C for 15 min, the reaction was initiated by adding 20 μL L-tyrosine solution (0.85 mM) to each well and incubating at room temperature for 10 min. The enzyme activity was determined by measuring the absorbance at 470 nm. Kojic acid was used as the positive control. The percent tyrosinase inhibition was calculated as follows: 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.

Xanthine Oxidase Inhibition Assay
Xanthine oxidase activity was determined by measuring the formation of uric acid from xanthine [41]. Phosphate buffer (300 μL, 50 mM, pH 7.4) was used in this assay. Varying concentrations of EO (100 μL) were mixed with a solution containing xanthine oxidase (100 μL, 0.2 U/mL) and water (100 μL). Samples were then incubated at 37 °C for 15 min. Xanthine (200 μL, 0.15 mM) was then added to each sample and further incubated at 37 °C for 30 min. The reaction was stopped by adding HCl (200 μL, 0.5 M).
The production of uric acid was determined by measuring the absorbance at 295 nm. The buffer was used as a blank, and the control was a solution containing xanthine and xanthine oxidase.
The percent xanthine oxidase inhibition was calculated by: Enzyme inhibition activity (%) = (1 − B/A) × 100 (4) where A is the enzyme activity without sample, and B is the activity in the presence of the sample.

Effects of Tairin and Shima EO on Melanin Biosynthesis in B16F10 Melanoma Cells
3.6.1. Cell Culture B16F10 melanoma cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (10,000 U/mL and 100 μg/mL) at 37 °C in a humidified atmosphere containing 5% CO2.

Cell Viability
Cell viability was determined after EO treatment using an MTT assay, as described by Campos et al. [42]. Briefly, B16F10 cells were plated at a density of 7 × 10 3 cells/well in a 96-well plate. After 48 h of culture, cells were exposed to varying concentrations of tairin and shima EOs (10, 50, and 100 μg/mL) or 500 μM kojic acid and incubated for an additional 48 h at 37 °C. Following incubation, the medium was removed, and the cells were washed twice with phosphate buffer and incubated with MTT solution (0.5 mg/mL) for 3 h at 37 °C. The medium was discarded, and 200 μL of ethanol was added. The absorbance of each well was measured at 570 nm using a microplate spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Measurement of Melanin Content
Melanin content was determined as described by Yoon et al. [43]. In brief, B16F10 cells were plated at a density of 7 × 10 3 cells/well in a 96-well plate. After 48 h of culture, cells were exposed to varying concentrations of tairin and shima EO (10, 20, and 50 μg/mL) or 500 μM kojic acid. After 1 h, 100 μM isobutyl-1-methylxanthine (IBMX) was added, and cells were incubated for an additional 48 h at 37 °C. The cells were washed twice with phosphate buffer, and then dissolved in 100 μL NaOH (1 N) containing 10% DMSO. Samples were incubated at 80 °C for 1 h and mixed to solubilize the melanin. The optical density of the mixed homogenate was measured at 490 nm. To measure the amount of melanin in each experiment, the total amount of melanin (100%) produced during the experimental period was considered as the control group, and the rate of inhibition in the treatment groups was calculated in proportion to this standard.

Intracellular Tyrosinase Activity
Tyrosinase activity was determined as described by Li et al. [44] with slight modifications. B16F10 cells were plated at a density of 7 × 10 3 cells/well in a 96-well plate. After 48 h of culture, cells were exposed to varying concentrations of tairin and shima EO (10, 20, and 50 μg/mL) or 500 μM kojic acid. After 1 h, 100 μM isobutyl-1-methylxanthine (IBMX) was added, and the cells were incubated for an additional 48 h at 37 °C. The cells then were washed with ice-cold phosphate buffer and lysed with phosphate buffer (pH 6.8) containing 1% Triton-X (90 μL/well). The plates were frozen at −80 °C for 30 min. After thawing and mixing, 10 μL of 1% L-DOPA was added to each well. Following incubation at 37 °C for 2 h, the absorbance was measured at 490 nm.

Statistical Analysis
Each experiment was performed in triplicate. All data are presented as the mean ± SE. Statistical analysis was done by one-way analysis of variance (ANOVA) complemented by the Duncan test. Differences were considered significant when the p value was less than 0.05.

Conclusions
This report is the first to demonstrate the anti-aging, anti-oxidant, and anti-melanogenesis activities of EOs from tairin and shima in cell culture. The present study reveals that EOs from tairin and shima have strong anti-oxidant activities against DPPH, ABTS, nitric oxide, singlet oxygen, and the hydroxyl radical, and inhibit xanthine oxidase. Tairin EO is a stronger inhibitor against a series of aging enzymes, such as collagenase, tyrosinase, hyaluronidase, and elastase, than shima EO. Moreover, both tairin and shima EOs are anti-melanogenic.