Antimelanogenic Effect of Urolithin A and Urolithin B, the Colonic

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Anti-melanogenic effect of urolithin A and urolithin B, the colonic metabolites of ellagic acid, in B16 melanoma cells Shang-Ta Wang, Wei-Chia Chang, Chen Hsu, and Nan-Wei Su J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02442 • Publication Date (Web): 20 Jul 2017 Downloaded from http://pubs.acs.org on July 21, 2017

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Journal of Agricultural and Food Chemistry

Anti-melanogenic effect of urolithin A and urolithin B, the colonic metabolites of ellagic acid, in B16 melanoma cells

Shang-Ta Wang, Wei-Chia Chang, Chen Hsu, Nan-Wei Su*

Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan

*Corresponding author: Nan-Wei Su, Ph.D., Professor Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan Tel: +886-2-33664819, Fax: +886-2-23632714 E-mail: [email protected]

Shang-Ta Wang, E-mail: [email protected]; Wei-Chia Chang, E-mail: [email protected]; Chen Hsu, E-mail: [email protected]

Keywords: urolithin; melanin; B16 melanoma cells; ellagic acid; tyrosinase

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ACS Paragon Plus Environment


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ABSTRACT

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Anti-melanogenic agents from natural sources have been widely investigated.

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Urolithin A (UA) and B (UB), the main gut microflora metabolites of dietary ellagic

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acid derivatives, have various bioactivities such as anti-inflammatory and anti-aging

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effects. In this study, the metabolites were found to possess depigmentation efficacy by

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suppressing tyrosinase activity. Both UA and UB could attenuate melanogenesis in B16

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melanoma cells to 55.1 ± 3.8 and 76.4 ± 17.4% of control at non-cytotoxic doses, 10

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µM, respectively. UA showed comparable efficacy to positive control, 5 µM ofkojic acid

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treatment (51.2 ± 7.8). RT-PCR results revealed that UA and UB inhibited melanin

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formation by affecting the catalytic activity of tyrosinase rather than its mRNA

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expression. Kinetics for UA and UB on tyrosinase activity revealed that their inhibition

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behavior toward cellular tyrosinase involved competitive inhibition. UA and UB may be

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potent tyrosinase inhibitors and they possess significant anti-melanogenesis ability as

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novel skin-whitening ingredients.

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Introduction

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The synthesis of melanin polymers in the melanosomes of melanocytes has been

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extensively studied 1. Inhibition of melanogenesis has been investigated in terms of

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reducing melanogenic intermediates, accelerating skin metabolism, and especially

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interfering in tyrosinase expression as well as inhibiting tyrosinase activity 2. Tyrosinase

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is the key enzyme catalyzing the oxidation of tyrosine to L-3, 4-dihydroxyphenylalanine

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(L-DOPA), L-DOPA to DOPA quinine, and 5, 6-dihydroxyindole to indole-5,6-quinone,

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thus leading to eumelanin formation in the absence of low-molecular-weight thiolic

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compounds such as glutathione and cysteine. Therefore, compounds targeting tyrosinase

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are of interest for developing skin-lightening agents 3, 4. The research on tyrosinase

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inhibition reveals that a large majority of the work has been carried out since 2000 and

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these compounds can be classified as competitive, uncompetitive, mixed type and

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non-competitive inhibitors. The nature of tyrosinase inhibition can be explored by

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measuring inhibition kinetics using Lineweaver-Burk plots with various concentrations

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of L-DOPA as the substrate. Knowledge of the classification of inhibition may be

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crucial to achieve better skin whitening effects since combination treatments may result

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in synergistic effects.

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Ellagic acid (4, 4′, 5, 5′, 6, 6′-hexahydroxydiphenic acid 2, 6, 2′, 6′-dilactone), the

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naturally occurring plant polyphenol abundant in berries and nuts, is produced from 3



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ellagitannins under hydrolytic conditions. Topical application of ellagic acid had

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depigmentation and photoprotective effects 5, 6. Shimogaki et al. concluded that the

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inhibitory effect of ellagic acid on melanogenesis was associated with its ability to

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chelate metallic ions of the tyrosinase molecule in melanocytes6. Yoshimura et al.

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suggested that uptake of foods rich in ellagic acid helped decrease melanoma

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differentiation and inhibit melanin biosynthesis 7. Nevertheless, ellagic acid was found

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with low plasma level and poor bioavailability on oral administration in healthy

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volunteers 8, 9; therefore, the role of dietary ellagic acid in melanogenesis in humans is

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controversial.

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Ellagitannins and ellagic acid are further metabolized by gut microbiota to urolithin A

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(UA) (3, 8-dihydroxy-6H-dibenzo[b,d]pyran-6-one) and urolithin B (UB)

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(3-dihydroxy-6H-dibenzo[b,d]pyran-6-one) (Fig. 1) after consumption and are

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biodistributed throughout plasma, organs, urine, and feces 10, 11. In humans, both

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ellagitannins and ellagic acid are poorly absorbed, whereas UA and UB show much

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higher plasma level, which suggests their greater biological potential than their parent

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compounds 11. These colonic metabolites of ellagic acid may play a role in

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hypomelanogenic activity.

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After consumption of ellagitannin-rich foods, these substances are primarily hydrolyzed into ellagic acid by tannase-producing bacteria or spontaneous hydrolysis in 4


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the gastrointestinal (GI) tract 12. Then ellagic acid is converted via lactone-ring opening,

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decarboxylation and further dehydroxylation enzymatic reactions into pentahydroxy

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urolithin (urolithin M5), tetrahydroxy urolithin (urolithin D, urolithin E and urolithin

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M6) by Gordonivater urolithincaciens and G. pamelaeae, the urolithin-producing

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colonic bacteria 13, 14. These multi-hydroxy urolithins are then transformed into urolithin

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C and finally form the major metabolites detected in human plasma, namely, UA and

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UB, by unknown bacterial dehydroxylation enzymes 15.

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UA and UB were proposed to be biomarkers and associated with the health effects of

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the consumption of ellagic acid–containing foods in humans 16. Recent study has

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revealed evidence of the biological effects of urolithins such as estrogenic,

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anti-estrogenic, anti-aging and anti-inflammatory effects, which supports their potential

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contribution to the health effects attributed to pomegranate and ellagitannin-rich foods

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17-19

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unexplored.

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. However, the effect of urolithins on melanogenesis in melanoma cells is still

We aimed to demonstrate the hypomelanogenic activity of UA and UB, the

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metabolites of ellagic acid, on melanin formation in murine B16F0 melanoma cells.

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Furthermore, we investigated their effects on the activity of tyrosinase in melanoma

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cells and the inhibition kinetics against tyrosinase activity.

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Material and Methods

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Chemicals

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UA and UB were from Kylolab (Ceuti, Spain). Murine B16F0 melanoma cell line

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(CRL-6322) was from ATCC (Manassas, VA, USA). Kojic acid, ascorbic acid, melanin,

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trypan blue, sodium hydroxide, dimethyl sulfoxide (DMSO), phenylmethanesulfonyl

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fluoride (PMSF), L-DOPA, Bovine Serum Albumin (BSA), mushroom tyrosinase,

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Triton X-100, Dulbecco modified Eagle medium (DMEM) and the salts for preparing

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phosphate buffer were from Sigma-Aldrich (St. Louis, MO, USA). Trypsin and

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antibiotic mixture solution were from Life Technologies Inc. (Grand Island, NY, USA).

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TRIzol reagent was from Invitrogen (Carlsbad, CA, USA). Bradford protein assay kit

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was from BIO-RAD Laboratories Inc. (Hercules, CA, USA). Fetal bovine serum (FBS)

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was from Gibco BRL (Gaithersburg, MD, USA). All chemicals were analytical grade

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and were from local suppliers in Taiwan.

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Cell culture B16F0 cells were maintained in DMEM containing 3.7 g/L sodium bicarbonate

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supplemented with 1% (w/v) nonessential amino acids, 10% (v/v) FBS and a 1% (v/v)

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penicillin (10,000 units/mL)–streptomycin (10 mg/mL) solution in a CO2 incubator. The

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cells were seeded at 3.0 × 105 cells/cm2 into T75 flasks and incubated at 37 °C under 6


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95% air–5% CO2 atmosphere until approximately 80% confluence, then harvested by

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trypsinization and seeded to a new flask until use.

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Cytotoxicity tests

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Toxicity of UA and UB to B16F0 cells was assessed by trypan blue dye uptake assay.

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Briefly, B16F0 cells were incubated in 12 mL DMEM with 10% FBS in T-75 flasks

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until 80% confluent, then subcultured in a 6-well plate at 2.5×105 cells per well in 2.4

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mL DMEM with 10% FBS. After 24 h of incubation, the medium was discarded and

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replaced with fresh DMEM containing 5% FBS and a urolithin sample at a final

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concentration of 5 to 30 µM. Each test was performed in triplicate and the final DMSO

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concentration in the medium was 0.4% as vehicle. The control group was incubated

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with only DMEM, with 5% FBS and 0.4% DMSO in the culture medium. After a

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further 72 h of incubation, cell viability was determined by trypan blue staining and

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cells were counted by using a hemocytocounter under a Nikon Eclipse TS100 inverted

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microscope. The highest concentration of tested samples that showed at least 80%

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survival of B16F0 cells was chosen for further experiments.

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Melanin quantification in B16F0 cells

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B16F0 cells were seeded in 6-well culture plates at 2.5 × 105 cells per well and

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cultured in 2.4 mL DMEM containing 10% FBS at 37 oC with 5% CO2 for 24 h. After

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replacing the culture medium with fresh DMEM supplemented with 5% FBS and 0.4%

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DMSO, cells were further incubated for 72 h to determine the constant production of

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melanin in cells as the control group. To determine the inhibitory effect of UA and UB

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on melanogenesis, the above fresh medium was replaced with medium containing UA

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or UB at 5 and 10 mM, with koji acid at 5 mM as a positive control. The final

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concentration of DMSO in medium for all treatments was 0.4%. After incubation for 72

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h, the medium was removed, and then the cells were washed twice with

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phosphate-buffered saline (PBS) and harvested by trypsinization with 0.25% trypsin in

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PBS. Melanin quantification was as described by Oka, M., et al. 20, with minor

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modification. Briefly, freshly trypsinized cells (2 × 106 cells) were pelleted by

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centrifugation at 1,000 × g for 5 min at 4°C, and then extracted with 0.4 mL of 1 N

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NaOH containing 10% DMSO at 80oC for 2 h and centrifuged at 11000 × g for 5 min.

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The absorbance of the supernatant was determined at 405 nm. The data are expressed as

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(%) melanin content calculated from differences in absorbance between control and

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urolithin-treated groups.

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Tyrosinase inhibition assay 8


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The procedures for the mushroom tyrosinase inhibition test were as described 21, with

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slight modification. Briefly, reaction mixtures were prepared by mixing 120 µL of 50

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mM phosphate buffer (pH 6.8), 4 µL of inhibitor solution, and 6 µL of 2,000 U/mL

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mushroom tyrosinase solution. After 10-min incubation, 70 µL of 1.5 mM L-DOPA was

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added to give final inhibitor concentrations of 5 µM for kojic acid, 100 µM for ascorbic

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acid, and 1, 5, 10, 25, 50, or 100 µM for urolithins. The mixture was then incubated for

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another 5 min at room temperature. UV absorbance was measured at 475 nm to

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determine dopachrome production.

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Furthermore, to examine the inhibitory effect of UA and UB on tyrosinase activity

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from B16F0 cells, tyrosinase extract was prepared as described 22. Briefly, B16F0 cells

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were harvested by trypsinization after 72-h culture and washed twice with PBS. Cell

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pellets were lysed with 100 µL lysis buffer containing 1% Triton X-100, 0.1 mM PMSF,

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and 50 mM PBS at pH 6.8. Cell lysates were then incubated at -80°C for 30 min and

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thawed at 4°C for 60 min to release tyrosinase from the melanosome membrane of cells.

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The suspension was centrifuged at 20,000 × g for 30 min at 4°C to separate melanin

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from cell lysates. The supernatant was collected and kept on ice for use as the enzyme

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extract. The protein content was determined by the Bio-Rad Bradford protein assay with

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BSA standards.

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For inhibition tests, 80 µL tyrosinase extract and 4 µL tested inhibitor solution was 9



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mixed in 96-well plates for 10 min. The catalysis reaction was then started by adding 40

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µL of 1.5 mM L-DOPA into the mixture. The final inhibitor concentrations were similar

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to those previously described. The mixture was mixed gently by using a vortex mixer

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for 5 sec each minute. Dopachrome formation in each well was determined after 10-min

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incubation at 37°C by measuring the UV absorbance at 475 nm with use of a microplate

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reader. The reaction mixture without L-DOPA substrate was used as a blank, and a

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vehicle control was also included. Inhibition of tyrosinase activity was calculated by the

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following equation (1).

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Inhibition (%) = (A－B)/A × 100

(1),

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where A is the difference in absorbance of the reaction mixture without the tested

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sample between samples with and without tyrosinase, and B is the difference in

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absorbance of the reaction mixture with the tested sample between samples with and

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without tyrosinase.

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The IC50 was then calculated by fitting the inhibition-concentration point with

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non-linear regression by using Graphpad Prism V5.0 (Graphpad PrismSoftware, San

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Diego, CA)

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Effect of UA and UB on mRNA expression of tyrosinase in B16F0 cells

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B16F0 cells were treated with 5 and 10 µM urolithin compounds for 72 h before

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RNA extraction from the harvested cells by the TRIzol reagent method. RNA samples

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(2 µg/reaction) were reverse-transcribed by using the SuperScript III platinum one-step

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quantitative RT-PCR system (Invitrogen, CA, USA) with oligo-dT and underwent

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reverse-transcription for amplification with Taq polymerase. The resulting cDNA was

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amplified with primers for mouse tyrosinase, sense,

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5’-GGCCAGCTTTCAGGCAGAGGT-3’ and antisense,

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5’-TGGTGCTTCATGGGCAAAATC-3’ and glyceraldehyde-3-phosphate

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dehydrogenase (GAPDH), sense, 5’-ACCACAGTCCATGCCATCAC-3’ and antisense,

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5’-TCCACCACCCTGTTGCTGTA-3’, as a normalization control. PCR thermal

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conditions were 94°C 30 sec, 70°C 30 sec, 72°C 40 sec with 33 cycles for tyrosinase

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and 94°C 30 sec, 65°C 30 sec, 72°C 40 sec with 28 cycles for GAPDH. The PCR

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products were separated by electrophoresis on 2.0% agarose gel containing ethidium

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bromide and quantified with use of Synoptics GeneTools 3.07 (Syngene, Cambridge,

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UK).

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Kinetics of B16F0 tyrosinase inhibition by UA and UB Kinetics analysis was as described 23. The reaction mixture consisted of 80 µL B16F0 11



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tyrosinase extract in PBS solution and UA or UB solution at pH 6.8. After the mixture

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was incubated at 37°C for 10 min, L-DOPA substrate solution at various concentrations

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was added into the reaction mixture. The concentration of inhibitors and substrate in the

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final mixture solution was 250 or 500 µM, and 0.75, 1.5, 3, and 6 mM, respectively. The

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absorbance at 475 nm was monitored in 1-min intervals for 20 min to determine the rate

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of dopachrome generation. The Michaelis constant (Km) and maximal velocity (Vmax) of

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tyrosinase and type of inhibition were determined by Lineweaver–Burk plots.

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Statistical analysis

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Independent Student's t test was used to compare the means of two groups. The level

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of significance was set at p < 0.05, p < 0.01 and P < 0.001. Statistical analyses involved

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use of SigmaPlot 10.0 (SPSS, inc., Chicago, IL, USA).

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Results and Discussion

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Cytotoxicity of UA and UB on B16F0 cells

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To investigate whether the urolithin compounds had significant cytotoxic effects on

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B16 cells, we tested a series of concentrations, 5 to 30 mM UA and UB, by trypan blue

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dye uptake assay to determine a feasible concentration for further studies. The threshold

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of cell viability for the maximal tolerable concentration was set to at least 80%, which 12


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was considered acceptable. Cell viability was reduced with increasing concentration of

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urolithins. Only 5 and 10 mM UA and UB were acceptable doses for further

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investigation of the anti-melanogenesis effect (Fig. 2).

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Effect of UA and UB on melanin formation In our study, the melanin content of B16-F10 melanoma cells without drug treatment

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was set to 100%. Without causing cytotoxicity, UA showed remarkable inhibition of

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melanin formation; the relative melanin content in B16F0 cells with 5 and 10 µM UA

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was 60.8% and 56.5%, respectively (Fig. 3), which was comparable to that of 5 µM

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kojic acid and better than that of 100 µM ascorbic acid, thereby indicating the high

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potential of UA to control skin pigmentation. UB also had an inhibitory effect on

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melanogenesis, attenuating the melanin formation to about 80% relative melanin

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content.

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Numerous inhibitors of melanin synthesis have been developed from natural sources,

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including polyphenols, alkaloids and curcuminoids 24. These active substances

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dose-dependently reduce the melanin content of B16 melanoma cells without cytotoxic

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effects and are therefore introduced into cosmetics as skin-whitening agents 25. As

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compared with the vehicle control group, quercetin attenuated cellular melanin

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production to 52% and 88% at 20 and 50 µM, respectively 26. Berberine at 30 µM 13



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inhibited 50% of melanin production, which was more effective than 200 µM arbutin 27.

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Curcumin had a signiﬁcant dose-dependent inhibitory eﬀect on melanin synthesis: the

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melanin content was 60%, 50% and 40% with 5, 7.5 and 10 µM curcumin 28. As a

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colonic metabolite of phytochemicals, UA showed a remarkable inhibition profile

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against melanin formation. The melanin content of B16F0 cells was reduced to nearly

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60% with only 5 µM UA, showing greater inhibitory efficiency than that of most natural

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source inhibitors 24. Therefore, UA may be a promising hypomelanogenesis agent.

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Consuming ellagitannin- and ellagic acid-rich food may help control skin

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pigmentation 6, 7. Nevertheless, these substances are practically not absorbed by the GI

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system 8, 9. In fact, the distribution and plasma concentration of urolithin metabolites

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must be addressed to assess the health benefits of ellagitannin- and ellagic

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acid-containing products, such as pomegranate juice and extract, strawberry, raspberry,

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walnut and oak-aged red wine. In previous study, the bioavailability and metabolic

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profiles of ellagitannins and ellagic acid were evaluated in healthy volunteers who

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consumed several foods and beverages in reasonable dietary quantities 29-32. The

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predominant metabolites found in human plasma were glucuronide conjugates of UA

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and UB 33, 34. These urolithins circulate in human plasma at 0.03 to 5.7 µM for

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UA-glucuronide and 0.012 to 7.3 µM for UB glucuronide, which were significantly

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higher than their parent compound, ellagic acid, which was only 0.04 to 0.07 µM in 14


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plasma 29-32. Therefore, with our study, ellgic acid-rich products may contribute their

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hypomelanogenesis function via UA and UB, demonstrated to have high potential in

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inhibiting melanin synthesis.

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Inhibitory effect of UA and UB on tyrosinases from mushroom and B16F0 cells

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Tyrosinase inhibitors have been of interest solely because of the key role of

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tyrosinase in catalyzing the first and only rate-limiting steps in melanogenesis.

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Therefore, the inhibition of tyrosinase has been intensively studied for screening

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depigmentation agents. First, we used mushroom tyrosinase (EC 1.14.18.1), which is

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similar to tyrosinases from other sources in both conformation and catalytic activities 35,

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to test the inhibitory effect of UA and UB. The positive controls, kojic acid at 5 µM and

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ascorbic acid at 100 µM, inhibited mushroom tyrosinase oxidizing L-DOPA by 14.2%

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and 9.9%, respectively (Fig. 4A). UA at beyond 5 µM showed comparable inhibition

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activity as ascorbic acid on mushroom tyrosinase activity. Meanwhile, UB at beyond 5

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µM exhibited significant inhibition of enzyme activity of greater than 30%, which was

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comparable to that with 5 µM kojic acid. The considerable inhibitory activity of

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cell-free tyrosinase by UA and UB suggests that these colonic metabolites may be

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potential substances for further study.

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Subsequently, we used the B16F0 melanoma-cell tyrosinase to further assess whether 15



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UA and UB would exhibit inhibitory activity of mammalian tyrosinase. Inhibition of the

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tested substance of cellular tyrosinase activity ranged from 18% to 52%, with dose

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dependent pattern along treatments (Fig. 4B), and both UA and UB possessed

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comparable inhibition activity to other previously used cosmetic agents at identical or

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lower levels. In addition, the IC50 of UA and UB was 19.2 and 25.1 µM toward

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mushroom tyrosinase; 83.3 and 91.6µM toward B16 cellular tyrosinas, respectively.

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Moreover, tested substances showed different inhibitory profiles between cell-free and

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cellular tyrosinase, from different origins. This finding may be attributed to the

271

differences in conformation of enzymes, catalytic rate, or stereo-hindrance by the

272

inhibitors. Similar findings were found for arbutin inhibiting mushroom and human

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tyrosinase activity 36. Hence, both UA and UB are potent tyrosinase inhibitors with

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stronger inhibitory effects on B16F0-cell than mushroom tyrosinase.

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Effect of UA and UB on tyrosinase gene expression in B16F0 cells

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Melanin formation can be inhibited by inhibiting tyrosinase translation or production,

278

directly inhibiting enzyme activity, or inhibiting the formation of melanin precursors or

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turning them into reduced forms 3. For example, piperlonguminine from Piper longum

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inhibited melanogenesis in B16 melanoma cells stimulated with the

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α-melanocyte–stimulating hormone 3-isobutyl-1-methylxanthine or with 16


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protoporphyrin IX by suppressing tyrosinase mRNA expression 37. However, arbutin,

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kojic acid and p-hydroxybenzyl alcohol directly inhibited the catalytic activity of

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tyrosinase without affecting gene expression 22, 38, 39, whereas ascorbic acid can reduce

285

the melanin precursors 40. The mechanism involved in melanogenic inhibition needs

286

more investigation for further utilization.

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In this study, we quantified the mRNA level of tyrosinase in B16F0 cells with the

288

treatments to determine whether the inhibitory effects on melanogenesis of UA and UB

289

involved in regulating tyrosinase gene expression. However, the mRNA expression did

290

not differ among all tested groups, and did not differ between urolithin-treated and

291

untreated cells (Fig. 5). Suppressing effects of UA and UB on melanin synthesis may be

292

attributed to their ability to interfere with B16F0 tyrosinase activity at the enzymatic but

293

not gene regulation level.

294 295

Kinetics of UA and UB inhibiting tyrosinase activity

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For enzyme kinetics, we used L-DOPA at various concentrations as the substrate and

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250 or 500 µM urolithins as the inhibitor to generate Lineweaver–Burk plots for B16F0

298

tyrosinase. Double reciprocal regression of 250 and 500 µM UA and the control resulted

299

in the same y intercept, so UA is a competitive inhibitor of melanocyte tyrosinase (Fig.

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6A). According to the reciprocal equation for competitive inhibition, 1/V = 1/Vmax + 17



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appKm/(Vmax [S]), we calculated the inhibition constant (Ki) of the competitive inhibitor

302

with the equation appKm = Km(1+([I]/Ki), where appKm is the apparent Km in the

303

presence of any inhibitor concentration. In the absence of inhibitors,

304

delta A475/min, with Km 5.02 mM. With 250 and 500 µM UA, the appKm of the

305

substrate increased to 10.72 and 27.03 mM, respectively. Thus, the Ki of UA was

306

estimated to be 114 µM. The inhibitory actions by UB on B16F0 tyrosinase also showed

307

the same y intercept in the Lineweaver–Burk plot (Fig. 6B). With 250 and 500 µM UB,

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the appKm of the substrate increased to 8.26 and 18.87 mM, respectively, with the Ki

309

estimated at 181 µM.

Vmax was 0.078

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Tyrosinase (EC 1.14.18.1) is a copper-containing mixed-function oxidase, widely

311

distributed in microorganisms, animals and plants. It is a tetramer composed of two H

312

subunits (43 kD) and two L subunits (13 kD) and contains two active sites, each

313

containing two copper atoms as the cofactors 41. The chemical structure of inhibitors is

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crucial for its inhibitory mechanisms by altering the ability to interrupt the enzymatic

315

catalysis reaction. Chang (2009) concluded that compounds with a coumarin backbone

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had multifunctional activity in skin care and great potential in tyrosinase inhibition 42.

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Some of these phenolics show good affinity for the enzyme, and the dopachrome

318

formation is therefore prevented. In terms of chemical structure, UA and UB both

319

possess coumarin backbones and do not seem to resemble any of the melanin precursors. 18


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The hypothesized mechanism of urolithins in our study may be interference of

321

substrates accessing the catalytic sites of the enzyme and therefore competitively

322

inhibiting tyrosinase activity, which agrees with literature reports.

323

In summary, our study revealed the inhibitory effect of UA and UB on tyrosinase

324

activity and melanin synthesis in murine B16F0 melanoma cells. The inhibitory

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mechanism may be competitive inhibition of tyrosinase activity rather than gene

326

regulation at the transcription level. UA and UB, the metabolites of ellagic acid, may

327

contribute to the whitening activity of ellagic acid-rich food and be potential agents for

328

controlling skin pigmentation. Therefore, further studies may be needed to assess the

329

potential for developing novel products of whitening agents by in vivo studies such as

330

rats or zebra fish43, and may have practical applications for humans.

331 332

Abbreviations

333

UA, urolithin A; UB, urolithin B; RT-PCR, reverse transcription-PCR; L-DOPA,

334

L-3,4-dihydroxyphenylalanine; GI, gastrointestinal; DMSO, dimethyl sulfoxide; PMSF,

335

phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; DMEM, Dulbecco's

336

Modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate buffered saline.

337 338

Acknowledgements 19



339

The authors would like to thank Dr. Ming-Tse Lin of Tatung University (Taipei, Taiwan)

340

for help with cell culture advising.

341 342

Funding Source

343

This work was a part of the research project, which was supported by the Ministry of

344

Science and Technology, Executive Yuan, Taiwan [grant number: MOST

345

101-2313-B-002-068-MY3].

346 347

Conflict of interest

348

The authors declare no competing financial interest.

349

20


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References

351

1. Sealy, R. C.; Hyde, J. S.; Felix, C. C.; Menon, I.; Prota, G., Eumelanins and

352

pheomelanins: characterization by electron spin resonance spectroscopy. Science

353

1982, 217, 545-547.

354

2. Pintus, F.; Matos, M. J.; Vilar, S.; Hripcsak, G.; Varela, C.; Uriarte, E.; Santana, L.;

355

Borges, F.; Medda, R.; Di Petrillo, A., New insights into highly potent tyrosinase

356

inhibitors based on 3-heteroarylcoumarins: Anti-melanogenesis and antioxidant

357

activities, and computational molecular modeling studies. Bioorganic & Medicinal

358

Chemistry 2017, 25, 1687-1695.

359

3. Parvez, S.; Kang, M.; Chung, H. S.; Cho, C.; Hong, M. C.; Shin, M. K.; Bae, H.,

360

Survey and mechanism of skin depigmenting and lightening agents. Phytotherapy

361

Research 2006, 20, 921-934.

362

4. Sapkota, K.; Roh, E.; Lee, E.; Ha, E.-M.; Yang, J.-H.; Lee, E.-S.; Kwon, Y.; Kim, Y.;

363

Na, Y., Synthesis and anti-melanogenic activity of hydroxyphenyl benzyl ether

364

analogues. Bioorganic & medicinal chemistry 2011, 19, 2168-2175.

365

5. Arima, M.; Nishizawa, H.; Takeuchi, K.; Deura, H.; Ishida, K., Agent containing an

366

ellagic acid series compound for external application and use thereof. In Google

367

Patents: 1991.

368

6. Shimogaki, H.; Tanaka, Y.; Tamai, H.; Masuda, M., In vitro and in vivo evaluation 21



369

of ellagic acid on melanogenesis inhibition. International journal of cosmetic

370

science 2000, 22, 291-304.

371

7. Yoshimura, M.; Watanabe, Y.; Kasai, K.; Yamakoshi, J.; Koga, T., Inhibitory effect

372

of an ellagic acid-rich pomegranate extract on tyrosinase activity and

373

ultraviolet-induced pigmentation. Bioscience, biotechnology, and biochemistry 2005,

374

69, 2368-2373.

375

8. Seeram, N. P.; Lee, R.; Heber, D., Bioavailability of ellagic acid in human plasma

376

after consumption of ellagitannins from pomegranate (Punica granatum L.) juice.

377

Clinica Chimica Acta 2004, 348, 63-68.

378

9. Hamad, A.-W. R.; Al-Momani, W. M.; Janakat, S.; Oran, S. A., Bioavailability of

379

ellagic acid after single dose administration using HPLC. Pakistan Journal of

380

Nutrition 2009, 8, 1661-1664.

381

10. Espín, J. C.; Larrosa, M.; García-Conesa, M. T.; Tomás-Barberán, F., Biological

382

significance of urolithins, the gut microbial ellagic acid-derived metabolites: the

383

evidence so far. Evidence-Based Complementary and Alternative Medicine 2013,

384

2013.

385

11. Zhang, W.; Chen, J. H.; Aguilera‐Barrantes, I.; Shiau, C. W.; Sheng, X.; Wang, L.

386

S.; Stoner, G. D.; Huang, Y. W., Urolithin A suppresses the proliferation of

387

endometrial cancer cells by mediating estrogen receptor‐α‐dependent gene 22


Page 22 of 37

Page 23 of 37


388

expression. Molecular nutrition & food research 2016, 60, 2387-2395.

389

12. Aguilar-Zárate, P.; Cruz-Hernandez, M.; Montañez, J.; Belmares-Cerda, R.; Aguilar,

390

C., Bacterial tannases: production, properties and applications. Revista Mexicana de

391

Ingeniería Química 2014, 13, 63-74.

392

13. Selma, M. V.; Beltrán, D.; García-Villalba, R.; Espín, J. C.; Tomás-Barberán, F. A.,

393

Description of urolithin production capacity from ellagic acid of two human

394

intestinal Gordonibacter species. Food & function 2014, 5, 1779-1784.

395

14. Selma, M. V.; Tomás-Barberán, F. A.; Beltran, D.; García-Villalba, R.; Espín, J. C.,

396

Gordonibacter urolithinfaciens sp. nov., a urolithin-producing bacterium isolated

397

from the human gut. International journal of systematic and evolutionary

398

microbiology 2014, 64, 2346-2352.

399

15. García-Villalba, R.; Beltrán, D.; Espín, J. C.; Selma, M. V.; Tomás-Barberán, F. A.,

400

Time course production of urolithins from ellagic acid by human gut microbiota.

401

Journal of agricultural and food chemistry 2013, 61, 8797-8806.

402

16. Cerdá, B.; Tomás-Barberán, F. A.; Espín, J. C., Metabolism of antioxidant and

403

chemopreventive ellagitannins from strawberries, raspberries, walnuts, and oak-aged

404

wine in humans: identification of biomarkers and individual variability. Journal of

405

agricultural and food chemistry 2005, 53, 227-235.

406

17. Larrosa, M.; González-Sarrías, A.; García-Conesa, M. T.; Tomás-Barberán, F. A.; 23



407

Espín, J. C., Urolithins, ellagic acid-derived metabolites produced by human colonic

408

microflora, exhibit estrogenic and antiestrogenic activities. Journal of Agricultural

409

and Food Chemistry 2006, 54, 1611-1620.

410

18. Sala, R.; Mena, P.; Savi, M.; Brighenti, F.; Crozier, A.; Miragoli, M.; Stilli, D.; Del

411

Rio, D., Urolithins at physiological concentrations affect the levels of

412

pro-inflammatory cytokines and growth factor in cultured cardiac cells in

413

hyperglucidic conditions. Journal of Functional Foods 2015, 15, 97-105.

414

19. Ryu, D.; Mouchiroud, L.; Andreux, P. A.; Katsyuba, E.; Moullan, N.;

415

Nicolet-dit-Félix, A. A.; Williams, E. G.; Jha, P.; Sasso, G. L.; Huzard, D., Urolithin

416

A induces mitophagy and prolongs lifespan in C. elegans and increases muscle

417

function in rodents. Nature Medicine 2016.

418

20.Oka, M.; Nagai, H.; Ando, H.; Fukunaga, M.; Matsumura, M.; Araki, K.; Ogawa, W.;

419

Miki, T.; Sakaue, M.; Tsukamoto, K., Regulation of melanogenesis through

420

phosphatidylinositol 3-kinase-Akt pathway in human G361 melanoma cells. Journal

421

of investigative dermatology 2000, 115, 699-703.

422

21. Itoh, K.; Hirata, N.; Masuda, M.; Naruto, S.; Murata, K.; Wakabayashi, K.; Matsuda,

423

H., Inhibitory effects of Citrus hassaku extract and its flavanone glycosides on

424

melanogenesis. Biological and Pharmaceutical Bulletin 2009, 32, 410-415.

425

22. Chung, S. W.; Ha, Y. M.; Kim, Y. J.; Song, S.; Lee, H.; Suh, H.; Chung, H. Y., 24


Page 24 of 37

Page 25 of 37


426

Inhibitory effects of 6-(3-hydroxyphenyl)-2-naphthol on tyrosinase activity and

427

melanin synthesis. Archives of pharmacal research 2009, 32, 289-294.

428

23. Hyun, S. K.; Lee, W.-H.; Jeong, D. M.; Kim, Y.; Choi, J. S., Inhibitory effects of

429

kurarinol, kuraridinol, and trifolirhizin from Sophora flavescens on tyrosinase and

430

melanin synthesis. Biological and Pharmaceutical Bulletin 2008, 31, 154-158.

431

24. Liu‐Smith, F.; Meyskens, F. L., Molecular mechanisms of flavonoids in melanin

432

synthesis and the potential for the prevention and treatment of melanoma. Molecular

433

nutrition & food research 2016, 60, 1264-1274.

434

25. Yoon, N. Y.; Eom, T.-K.; Kim, M.-M.; Kim, S.-K., Inhibitory effect of phlorotannins

435

isolated from Ecklonia cava on mushroom tyrosinase activity and melanin

436

formation in mouse B16F10 melanoma cells. Journal of agricultural and food

437

chemistry 2009, 57, 4124-4129.

438

26. Yang, Y. M.; Son, Y. O.; Lee, S. A.; Jeon, Y. M.; Lee, J. C., Quercetin inhibits

439

α‐MSH‐stimulated melanogenesis in B16F10 melanoma Cells. Phytotherapy

440

Research 2011, 25, 1166-1173.

441

27. Song, Y. C.; Lee, Y.; Kim, H. M.; Hyun, M. Y.; Lim, Y. Y.; Song, K. Y.; Kim, B. J.,

442

Berberine regulates melanin synthesis by activating PI3K/AKT, ERK and GSK3β in

443

B16F10 melanoma cells. International journal of molecular medicine 2015, 35,

444

1011-1016. 25



445

28. Tu, C. X.; Lin, M.; Lu, S. S.; Qi, X. Y.; Zhang, R. X.; Zhang, Y. Y., Curcumin

446

inhibits melanogenesis in human melanocytes. Phytotherapy Research 2012, 26,

447

174-179.

448

29. Cerdá, B.; Espín, J. C.; Parra, S.; Martínez, P.; Tomás-Barberán, F. A., The potent in

449

vitro antioxidant ellagitannins from pomegranate juice are metabolised into

450

bioavailable but poor antioxidant hydroxy–6H–dibenzopyran–6–one derivatives by

451

the colonic microflora of healthy humans. European journal of nutrition 2004, 43,

452

205-220.

453

30.González-Sarrías, A.; García-Villalba, R.; Núñez-Sánchez, M. Á.; Tomé-Carneiro, J.;

454

Zafrilla, P.; Mulero, J.; Tomás-Barberán, F. A.; Espín, J. C., Identifying the limits for

455

ellagic acid bioavailability: A crossover pharmacokinetic study in healthy volunteers

456

after consumption of pomegranate extracts. Journal of Functional Foods 2015, 19,

457

225-235.

458

31. Natella, F.; Leoni, G.; Maldini, M.; Natarelli, L.; Comitato, R.; Schonlau, F.; Virgili,

459

F.; Canali, R., Absorption, metabolism, and effects at transcriptome level of a

460

standardized French oak wood extract, Robuvit, in healthy volunteers: pilot study.

461

Journal of agricultural and food chemistry 2014, 62, 443-453.

462

32. Seeram, N. P.; Henning, S. M.; Zhang, Y.; Suchard, M.; Li, Z.; Heber, D.,

463

Pomegranate juice ellagitannin metabolites are present in human plasma and some 26


Page 26 of 37

Page 27 of 37


464

persist in urine for up to 48 hours. The Journal of Nutrition 2006, 136, 2481-2485.

465

33. Tomás-Barberán, F. A.; García-Villalba, R. o.; González-Sarrías, A.; Selma, M. V.;

466

Espín, J. C., Ellagic acid metabolism by human gut microbiota: consistent

467

observation of three urolithin phenotypes in intervention trials, independent of food

468

source, age, and health status. Journal of agricultural and food chemistry 2014, 62,

469

6535-6538.

470

34. García-Muñoz, C.; Hernández, L.; Pérez, A.; Vaillant, F., Diversity of urinary

471

excretion patterns of main ellagitannins' colonic metabolites after ingestion of

472

tropical highland blackberry (Rubus adenotrichus) juice. Food Research

473

International 2014, 55, 161-169.

474 475

35. Chen, Q.-X.; Kubo, I., Kinetics of mushroom tyrosinase inhibition by quercetin. Journal of agricultural and food chemistry 2002, 50, 4108-4112.

476

36. Maeda, K.; Fukuda, M., Arbutin: mechanism of its depigmenting action in human

477

melanocyte culture. Journal of Pharmacology and Experimental Therapeutics 1996,

478

276, 765-769.

479

37. Kim, K. S.; Kim, J. A.; Eom, S. Y.; Lee, S. H.; Min, K. R.; Kim, Y., Inhibitory effect

480

of piperlonguminine on melanin production in melanoma B16 cell line by

481

downregulation of tyrosinase expression. Pigment cell research 2006, 19, 90-98.

482

38. Liu, X.; Osawa, T., Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher 27



483

antioxidant activity in vitro compared to the all-trans isomer. Biochemical and

484

biophysical research communications 2007, 357, 187-193.

485

39. Battaini, G.; Monzani, E.; Casella, L.; Santagostini, L.; Pagliarin, R., Inhibition of

486

the catecholase activity of biomimetic dinuclear copper complexes by kojic acid.

487

Journal of Biological Inorganic Chemistry 2000, 5, 262-268.

488

40. Tsuji-Naito, K.; Hatani, T.; Okada, T.; Tehara, T., Modulating effects of a novel

489

skin-lightening agent, α-lipoic acid derivative, on melanin production by the

490

formation of DOPA conjugate products. Bioorganic & medicinal chemistry 2007, 15,

491

1967-1975.

492

41. Yong, G.; Leone, C.; Strothkamp, K. G., Agaricus bisporus metapotyrosinase:

493

preparation, characterization, and conversion to mixed-metal derivatives of the

494

binuclear site. Biochemistry 1990, 29, 9684-9690.

495 496

42. Chang, T.-S., An updated review of tyrosinase inhibitors. International journal of molecular sciences 2009, 10, 2440-2475.

497

43. Hsu, K. D.; Chen, H. J.; Wang, C. S.; Lum, C. C.; Wu, S, P,; Lin, S. P.; Cheng, K. C.,

498

Extract of Ganoderma formosanum mycelium as a highly potenttyrosinase inhibitor.

499

Scientific Report 2016, 6, 32584.

500

28


Page 28 of 37

Page 29 of 37


501

Figure Caption

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Fig. 1. Chemical structures of (A) urolithin A and (B) urolithin B.

503 504

Fig. 2. Viability of B16F0 melanoma cells treated with various concentrations of

505

urolithins. Dotted line is 80% acceptable tolerance. Data are mean ± SD (n=3).

506 507

Fig. 3. (A) Inhibitory effect of urolithin A and urolithin B on melanin formation in

508

B16F0 cells. Kojic acid and ascorbic acid were positive controls. (B) Images of

509

accumulation of melanin in living and harvested cells. Data are mean ± SD (n=3).

510

*p

Antimelanogenic Effect of Urolithin A and Urolithin B, the Colonic

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