Anti-melanogenic 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.

275 276

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

297

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.

300

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,

308

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

310

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

314

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.

317

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

325

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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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

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*p

Anti-melanogenic effect of urolithin A and urolithin B, the colonic

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