Antityrosinase, Antioxidant, and Cytotoxic Activities of Phytochemical Constituents from Manilkara zapota L. Bark

Hyperpigmentation is considered by many to be a beauty problem and is responsible for photoaging. To treat this skin condition, medicinal cosmetics containing tyrosinase inhibitors are used, resulting in skin whitening. In this study, taraxerol methyl ether (1), spinasterol (2), 6-hydroxyflavanone (3), (+)-dihydrokaempferol (4), 3,4-dihydroxybenzoic acid (5), taraxerol (6), taraxerone (7), and lupeol acetate (8) were isolated from Manilkara zapota bark. Their chemical structures were elucidated by analysis of their nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) data, and by comparing them with data found in the literature. The in vitro antityrosinase, antioxidant, and cytotoxic activities of the isolated compounds (1–8) were evaluated. (+)-Dihydrokaempferol (4) exhibited higher monophenolase inhibitory activity than both kojic acid and α-arbutin. However, it showed diphenolase inhibitory activity similar to kojic acid. (+)-Dihydrokaempferol (4) was a competitive inhibitor of both monophenolase and diphenolase activities. It exhibited the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) activities of the isolated compounds. Furthermore, (+)-dihydrokaempferol (4) also demonstrated potent cytotoxicity in breast carcinoma cell line (BT474), lung bronchus carcinoma cell line (Chago-K1), liver carcinoma cell line (HepG2), gastric carcinoma cell line (KATO-III), and colon carcinoma cell line (SW620). These results suggest that M. zapota bark might be a good potential source of antioxidants and tyrosinase inhibitors for applications in cosmeceutical products.


Introduction
It is well known that free radicals constitute a major risk factor for many diseases, such as cancer, hypertension, asthma, diabetes, and Alzheimer's disease, as well as aging [1,2]. Antioxidant activity is the most important property of phytochemicals that prevent cellular molecules from oxidative stress. Secondary metabolites of plants are natural antioxidants [3,4]. Antioxidants such as phenolic compounds, flavonoids, and polyphenols trap free radicals and inhibit oxidative stress mechanisms. The oxidation of free radicals can cause the occurrence of melanoma and cancer [5,6]. In the skin, ultraviolet radiation induces the generation of reactive oxygen species (ROS). The ROS mechanism accumulates skin pigmentation on melanocytes. Then, ROS accelerate epidermal phenylalanine hydroxylase (PAH; EC 1.14. 16.1). PAH is the rate-limiting enzyme for the production of l-tyrosine. l-Tyrosine is the initial substrate of tyrosinase [7]. Tyrosinase or polyphenol oxidase (EC 1.14.18.1, PPO) is a copper-containing enzyme in melanin biosynthesis [8]. Tyrosinase catalyzes the * Kojic acid and α-arbutin were used as positive controls. Each value represents the mean ± standard deviation of three independent replicates. Different letters in the same column indicate significant differences (p < 0.05) within conditions according to Tukey's multiple range Test.

Kinetic Inhibition of Compounds 1-8 on Tyrosinase Inhibitory Activity
The kinetic inhibition of compounds 1-8 was determined with respect to both monophenolase and diphenolase activities (Figures 2 and 3). The double-reciprocal plots of 1/V versus 1/[S] showed straight lines with individual slopes and the same horizontal-axis intercept (Figure 2a,b,f). The results indicate that the inhibitor affected the velocity of reaction, but it did not affect the enzyme-substrate complex. It was determined that taraxerol methyl ether (1), spinasterol (2), and taraxerol (6)  The results demonstrate that taraxerone (7) and lupeol acetate (8) were mixed inhibitors.
With respect to diphenolase inhibitory activity (Figure 3), taraxerol methyl ether (1) and spinasterol (2) were noncompetitive inhibitors. Their Lineweaver-Burk plots show a family of lines with different slopes in which the V max values were altered, whereas the K m value persisted with the increasing concentration of the inhibitors (Figure 3a,b). 6-Hydroxyflavanone (3), (+)-dihydrokaempferol (4), and 3,4-dihydroxybenzoic acid (5) inhibited diphenolase activity in a competitive manner. The values of K m enlarged with the increase of the inhibitors' concentration, and the value of V max did not change (Figure 3c-e). Taraxerol (6) and lupeol acetate (8) were uncompetitive inhibitors. Their Lineweaver-Burk plots (Figure 3f,h) show that both the V max and K m values were altered with the increasing concentration of taraxerol (6) and lupeol acetate (8). The relationship between plots of 1/V and 1/[S] of taraxerone (7) show a family of straight lines that intersect on the left of the vertical axis ( Figure 3g). The results demonstrate that taraxerone (7) was a mixed inhibitor. This is because taraxerone (7) can bind to free enzymes as well as to enzyme-substrate complexes.  (6); (g) taraxerone (7); and (h) lupeol acetate (8).
With respect to diphenolase inhibitory activity (Figure 3), taraxerol methyl ether (1) and spinasterol (2) were noncompetitive inhibitors. Their Lineweaver-Burk plots show a family of lines with different slopes in which the Vmax values were altered, whereas the Km value persisted with the competitive manner. The values of Km enlarged with the increase of the inhibitors' concentration, and the value of Vmax did not change (Figure 3c-e). Taraxerol (6) and lupeol acetate (8) were uncompetitive inhibitors. Their Lineweaver-Burk plots (Figure 3f,h) show that both the Vmax and Km values were altered with the increasing concentration of taraxerol (6) and lupeol acetate (8). The relationship between plots of 1/V and 1/[S] of taraxerone (7) show a family of straight lines that intersect on the left of the vertical axis (Figure 3g). The results demonstrate that taraxerone (7) was a mixed inhibitor. This is because taraxerone (7) can bind to free enzymes as well as to enzyme-substrate complexes.  (6); (g) taraxerone (7); and (h) lupeol acetate (8).
Furthermore, the kinetic inhibition demonstrated by compounds 1-8 occurred in a dose-dependent manner. The inhibition of 6-hydroxyflavanone (3), (+)-dihydrokaempferol (4), and 3,4-dihydroxybenzoic acid (5) was shown to be competitive inhibition of both monophenolase and diphenolase activities. This indicates that competitive inhibitors only bind with free enzymes [50]. Additionally, it has previously been reported that 3,4-dihydroxybenzoic acid (5) is also a tyrosinase substrate, but that its K m is lower than l-DOPA; consistent with its characterization as a competitive inhibitor [53]. Taraxerol methyl ether (1) and spinasterol (2) were determined to be noncompetitive inhibitors with respect to both monophenolase and diphenolase activities. Based on the results, we found that noncompetitive inhibitors depend on the velocity of reaction and bind at different sites on enzymes. Taraxerol (6) was a noncompetitive inhibitor with respect to monophenolase inhibitory activity, but it was an uncompetitive inhibitor with respect to diphenolase inhibitory activity. An uncompetitive inhibitor binds only to enzyme-substrate complexes. Taraxerone (7) was a mixed inhibitor with respect to both monophenolase and diphenolase activities. These results indicate that taraxerone (7) did not bind to the active sites of enzymes. Previous research reported that the inhibitory mechanism is based on several factors, such as the ability to engage in copper chelating, lack of free radical scavenging, and binding of a compound to the active site of an enzyme [54]. Lupeol acetate (8) was a mixed inhibitor with respect to monophenolase inhibitory activity, but it was an uncompetitive inhibitor of diphenolase inhibitory activity. A mixed inhibitor binds to free enzymes and enzyme-substrate complexes at separate sites that are not active sites, whereas an uncompetitive inhibitor binds to enzyme-substrate complexes at separate sites but does not bind to free enzymes [54].
In this study, 6-hydroxyflavanone (3), (+)-dihydrokaempferol (4), and 3,4-dihydroxybenzoic acid (5) were potent antioxidants. Usually, antioxidants can protect an organism against ROS. 6-Hydroxyflavanone (3) and (+)-dihydrokaempferol (4) are flavonoids, which can donate hydrogen similar to phenolic compounds, such as 3,4-dihydroxybenzoic acid (5). Thus, flavonoids possess free radical scavenging abilities similar to phenolic compounds [55]. The results demonstrated that the compounds with more phenolic hydroxyl groups have more antityrosinase and antioxidant activities. Therefore, these compounds might have the potential to be used in treatment of skin depigmentation via inhibition of tyrosinase activity. Interestingly, (+)-dihydrokaempferol (4) also displayed potent cytotoxicity in the carcinoma cell lines tested: BT474, Chago-K1, HepG2, KATO-III, and SW620. Moreover, it was not toxic to the normal cell line, WI-38. These results suggest that (+)-dihydrokaempferol (4) might have the potential to be a good candidate for treatment of skin depigmentation. Thus, M. zapota bark might be a good potential source of antioxidants and tyrosinase inhibitors for applications in cosmeceutical products. Additionally, the results of this work may be useful in the study of the structure-activity relationships of flavonoids and antityrosinase activity, to guide the synthesis of desirable new compounds which can act as potent tyrosinase inhibitors.

Plant Material
The bark of M. zapota was collected from Saraburi Province, Thailand, in May 2013. The voucher specimen Bangkok forest herbarium No. 187749 (BKF No. 187749) was deposited at the Forest Herbarium Department of National Parks, Wildlife, and Plant Conservation, Bangkok, Thailand.

Mushroom Tyrosinase Inhibitory Assay
The inhibition of tyrosinase activity was performed by spectrophotometry using a modified method of a previously described procedure [56]. l-Tyrosine and l-DOPA were used as substrates for monophenolase and diphenolase activity, respectively. Briefly, a sample was dissolved in a mixture of DMSO/ethanol (1:4 v/v). The reaction mixture consisted of 150 µL of 0.2 M sodium phosphate buffer (pH 6.8), 50 µL of sample, and 50 µL of substrate solution (500 µM for l-tyrosine/l-DOPA). The reaction was mixed and was incubated for 10 min at 30 • C. Then, 50 µL of tyrosinase solution (200 U/mL) was added, and absorbance was immediately measured at 490 nm (t = 0 min). The assay mixture was then incubated for 20 min at 30 • C, and absorbance was measured at 490 nm (t = 20 min). Kojic acid and α-arbutin were used as positive controls. The percentage of inhibition of tyrosinase activity was calculated using the following equation: where A is the difference of the absorbance of the control at t = 0 min and t = 20 min, B is the difference of the absorbance of the blank control at t = 0 min and t = 20 min, C is the difference of the absorbance of the test sample and the positive control at t = 0 min and t = 20 min, and D is the difference of the absorbance of the blank of the test sample and the positive control at t = 0 min and t = 20 min.

Kinetic Analysis of Tyrosinase Inhibitory Activity
The kinetic analysis of tyrosinase inhibitory activity was performed with respect to both monophenolase and diphenolase activities. The concentration ranges of the samples were 20-1000 µM. Both l-tyrosine and l-DOPA were concentrated at 0, 25, 50, 100, and 200 µM, respectively. The inhibitory kinetics of the samples were analyzed using Lineweaver-Burk plots.

DPPH Radical Scavenging Assay
The DPPH radical scavenging activity was determined by a modified method based on a previously described procedure [57]. Briefly, a solution containing 50 µL of sample (100 mg/mL) was dissolved in DMSO/ethanol (1:4 v/v) and 150 µL of 0.05 M DPPH solution in methanol. Then, the reaction mixture was mixed and was incubated in the dark for 30 min at 37 • C. The absorbance of the reaction mixture was measured at 517 nm. Trolox was used as a positive control. The DPPH scavenging effect was calculated according to the following equation: where A s is the absorbance of the sample mixed with DPPH solution, A b is the absorbance of the sample without DPPH solution, and A d is the absorbance of DPPH solution without the sample.

ABTS Radical Scavenging Assay
The ABTS radical scavenging capacity was determined using a modified version of a previously described procedure [58]. The stock solution included 100 mL of 7.0 mM ABTS solution in methanol and 100 mL of 2.4 mM aqueous solution of potassium persulfate. Then, the reaction mixture was left in the dark for 14 h at 37 • C. The solution of 1 mL of ABTS solution was diluted with 60 mL of absolute ethanol to determine an absorbance of 0.700 ± 0.001 units at 734 nm using a spectrophotometer. Next, 500 µL of the sample (100 mg/mL) was reacted with 500 µL of ABTS solution and the absorbance was measured at 734 nm after 7 min of incubation using a spectrophotometer. The results were compared with Trolox as a standard, and the percentage of scavenging activity was calculated according to the following equation: where A c is the absorbance of ABTS radicals with ethanol and A s is the absorbance of ABTS radicals with the test sample or positive control.

FRAP Assay
FRAP assay was conducted using a modified version of a method originally reported in an earlier study [58]. The FRAP reagent contained 25 mL of 0.3 M acetate buffer (pH 3.6), 2.5 mL of 20 mM ferric chloride solution, and 2.5 mL of 10 mM 2,4,6-tris(2-pyridyl)-1,3,5-triazine and was brought to a final volume of 50 mL using 40 mM HCl solution. Then, the FRAP reagent was put into a water bath for 30 min at 50 • C. Next, 600 µL of FRAP reagent was added to 25 µL of the sample (100 mg/mL). The absorbance was recorded at 595 nm after 4 min of incubation using a spectrophotometer. Trolox was used as a positive control. The ferric reducing capacity was expressed as a ferrous sulphate equivalent.

Cytotoxicity Assay
Cytotoxic activity was evaluated in vitro using the microtitration colorimetric method of MTT reduction [59]. In this study, five human carcinoma cell lines were used, including BT474 (ATCC ® HTB20 TM ), Chago-K1 (National Cancer Institute, Thailand), HepG2 (ATCC ® HB8065 TM ), KATO-III (ATCC ® HTB103 TM ), and SW620 (ATCC ® CCL227 TM ). Additionally, human diploid lung fibroblast (WI-38, ATCC ® CCL75 TM ) was used as the normal cell line for comparison with the carcinoma cell lines. The culturing of these cell lines was derived in complete medium, including Roswell Park Memorial Institute medium (RPMI-1640), fetal bovine serum (5%, v/v), 25 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium bicarbonate (0.25%, w/v), and kanamycin (100 µg/mL). Doxorubicin was used as a positive control. Each well plate contained 198 µL of culture medium of cell lines and was incubated with 5% CO 2 atmosphere for 24 h at 37 • C. Then, the culture cells were treated with 2 µL/well of the sample and incubated for 72 h at 37 • C. MTT solution (2 µL, 5 mg/mL in normal saline) was added into each well, and the plates were incubated for an additional 4 h. The supernatant was aspirated out. After that, a mixture of 25 µL of 0.1 M glycine and 150 µL of DMSO was added. The plates were shaken to dissolve the purple-blue crystal of formazan. Then, the absorbance was determined by a microplate reader at 540 nm. The relative cell survival as a percentage of the control (DMSO), which was set at 100%, was calculated using the following formula: The cell survival (%) = A s A c × 100.
where A s is the absorbance of the test sample and A c is the absorbance of a positive control.

Statistical Analysis
All experiments were repeated in triplicate. All data are expressed as mean ± standard deviation. Statistical analyses were evaluated by GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Differences between treatments means were separated by the Tukey test at a significance level of p < 0.05.
Funding: This research was funded by the Graduate School of Chulalongkorn University (Thesis Grant), Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (GRU 6203023003-1) and the National Research Council of Thailand (GRB_BSS_101_59_61_08).