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Correlation between the Potency of Flavonoids on Mushroom Tyrosinase Inhibitory Activity and Melanin Synthesis in Melanocytes Worrawat Promden 1, * ID , Wittawat Viriyabancha 2 , Orawan Monthakantirat 3 , Kaoru Umehara 4,5 , Hiroshi Noguchi 4,6 and Wanchai De-Eknamkul 7, * 1 2 3 4 5 6 7

*

Division of General Science, Faculty of Education, Buriram Rajabhat University, Buriram 31000, Thailand Bureau of Drug Control, Food and Drug Administration, Ministry of Public Health, Nonthaburi 11000, Thailand; [email protected] Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; [email protected] School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan; [email protected] (K.U.); [email protected] (H.N.) Faculty of Pharmaceutical Sciences, Yokohama University of Pharmacy, Yokohama, Kanagawa 245-0066, Japan Department of Pharmacognosy, Nihon Pharmaceutical University, Kitaadachi, Saitama 362-0806, Japan Natural Product Biotechnology Research Unit, Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Correspondences: [email protected] (W.P.); [email protected] (W.D.-E.); Tel.: +66-89-424-2324 (W.P.); +66-2-218-8393 (W.D.-E.)

Received: 6 May 2018; Accepted: 8 June 2018; Published: 9 June 2018







Abstract: Twenty-seven flavonoids isolated from Dalbergia parviflora with vast structural diversity were screened for inhibitory activity against mushroom and murine tyrosinases using L -DOPA as the substrate. Among the flavonoids tested, only four—khrinone (5), cajanin (9), (3RS)-30 -hydroxy-8-methoxy vestitol (21), and (6aR,11aR)-3,8-dihydroxy-9-methoxy pterocarpan (27)—reacted with mushroom tyrosinase, with IC50 values of 54.0, 67.9, 67.8, and 16.7 µM, respectively, and only compound 27 showed inhibitory activity against murine tyrosinase. With cell-based assays, only compounds 9 and 27 effectively inhibited melanogenesis in B16-F10 melanoma cells (by 34% and 59%, respectively), at a concentration of 15 µM, without being significantly toxic to the cells. However, the crude extract of D. parviflora and some of the flavonoid constituents appeared to increase melanin production in B16-F10 cells, suggesting that there are flavonoids with both inhibitory and stimulatory melanogenesis in the crude extract. Studies on the correlation between the enzyme-based and cell-based assays showed that only the flavonoids with IC50 values below 50 µM against mushroom tyrosinase could inhibit the mammalian tyrosinase, and thus, reduce melanogenesis in B16-F10. Flavonoids with the IC50 values greater than 50 µM, on the other hand, could not inhibit the mammalian tyrosinase, and had either no effect or enhancement of melanogenesis. In conclusion, the tyrosinase enzyme from mushroom is not as selective as the one from mammalian source for the enzyme-based melanogenesis inhibitory screening, and the mammalian cell-based assay appears to be a more reliable model for screening than the enzyme-based one. Keywords: tyrosinase; flavonoid; Dalbergia parviflora; melanogenesis

1. Introduction Tyrosinase (EC 1.14.18.1) is the key enzyme involved in melanin biosynthesis in living organisms. In animal cells, tyrosinase oxidizes L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA), Molecules 2018, 23, 1403; doi:10.3390/molecules23061403

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and subsequently, L-DOPA to DOPA quinone, in the initial steps in melanogenesis, a process which is Molecules 2017, 22, x FOR PEER REVIEW 2 of 11 mainly responsible for dark skin color. In plant-derived foods, tyrosinase catalyzes the oxidation of phenolic compounds to their corresponding quinones, and is responsible for the enzymatic browning Tyrosinase (EC 1.14.18.1) is the key enzyme involved in melanin biosynthesis in living organisms. of fruits and vegetables [1]. Therefore, tyrosinase inhibitors have become increasingly important in the In animal cells, tyrosinase oxidizes L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA), and cosmetic field as skin-whitening agents and in the food industry [2–5]. subsequently, L-DOPA to DOPA quinone, in the initial steps in melanogenesis, a process which is Mushroom tyrosinase widely used as afoods, substitute for mammalian mainly responsible for darkhas skinbeen color. In plant-derived tyrosinase catalyzes the tyrosinase oxidation ofto screen for phenolic tyrosinase inhibitors as it is inexpensive and commercially available in a compounds to their corresponding quinones, and is responsible for the enzymaticpurified browningform [5]. Several plant have been reported to inhibitors have varying degreesincreasingly of inhibitory activity of fruits andflavonoids vegetables [1]. Therefore, tyrosinase have become important in towards the cosmetic field as skin-whitening agents in the foodinhibitors industry [2–5]. mushroom tyrosinase, and are thought to and be potential of melanin synthesis in mammalian Mushroom tyrosinase has been widely as a substitute for mammalian to screen between melanocytes [6–8]. However, there is a used limitation of information abouttyrosinase the correlation for tyrosinase inhibitors as it is inexpensive and commercially available in a purified form [5]. Several the inhibitory activity of mushroom tyrosinase by flavonoids and the actual inhibitory effect of plant flavonoids have been reported to have varying degrees of inhibitory activity towards melanin formation in mammalian cells. Only one report has indicated that the inhibition rate mushroom tyrosinase, and are thought to be potential inhibitors of melanin synthesis in mammalian of mushroom tyrosinase might not the inhibition rate of melanin in melanocytes [6–8]. However, there is aaccurately limitation ofreflect information about the correlation betweensynthesis the melanocytes [9]. We have reported, the structure ofmelanin estrogenic-like inhibitory activity of mushroom tyrosinasepreviously, by flavonoids andisolation the actual and inhibitory effect of compounds from the heartwood ofone Dalbergia parviflora, Thaithe folk medicine formation in mammalian cells. Only report has indicateda that inhibition rate traditionally of mushroom used as tyrosinase accurately reflect the inhibition rate of melanin synthesiswere in melanocytes a blood tonicmight and not menstruation normalizer. More than 60 flavonoids isolated [9]. andWe identified, have reported, previously, the isolation and structure of estrogenic-like compounds from the and six including 22 isoflavones, 12 isoflavanones, 10 isoflavans, six flavonones, four flavanonols, heartwood of Dalbergia parviflora, a Thai folk medicine traditionally used as a blood tonic and pterocarpans [10,11]. The structure–antioxidant relationship in the isoflavonoids from D. parviflora menstruation normalizer. More than 60 flavonoids were isolated and identified, including 22 has also been reported [12]. The structural diversity of the flavonoids has provided a good source for isoflavones, 12 isoflavanones, 10 isoflavans, six flavonones, four flavanonols, and six pterocarpans studying between enzymatic cell-based assays. [10,11].the Thecorrelation structure–antioxidant relationshipassay in the and isoflavonoids from D. parviflora has also been In this [12]. study, extract and of 27the flavonoids anda isolated from heartwood of D. reported Thecrude structural diversity flavonoidsprepared has provided good source for the studying the parviflora werebetween tested enzymatic for their ability to inhibit mushroom and murine tyrosinases using L-DOPA as correlation assay and cell-based assays. In this study, crude extract and 27 flavonoids prepared and isolated from the of D. of crude the substrate. The inhibition of mushroom tyrosinase was then compared toheartwood the inhibition parviflora were tested for their ability to inhibit mushroom and murine tyrosinases using L -DOPA as murine tyrosinase extracted from B16-F10 melanoma cells. Finally, the effects of these compounds on the substrate. The inhibition of mushroom tyrosinase was then compared to the inhibition of crude the overall melanin production in B16-F10 melanoma cells were studied to observe the correlation murine tyrosinase extracted from B16-F10 melanoma cells. Finally, the effects of these compounds on between the results of the enzyme-based and cell-based assays. the overall melanin production in B16-F10 melanoma cells were studied to observe the correlation between the results of the enzyme-based and cell-based assays.

2. Results and Discussion

2. Results and Discussion

2.1. Inhibitory Effect of Flavonoids on the o-Diphenolase Activity of Mushroom Tyrosinase 2.1. Inhibitory Effectofofthe Flavonoids on the o-Diphenolase Activity of Mushroom Tyrosinase The structures 27 flavonoids isolated from D. parviflora, grouped into isoflavones, flavanones,

isoflavanones, isoflavans, pterocarpan, are shown Table 1. grouped Each compound (200 µM) was The structures of theand 27 flavonoids isolated from D.inparviflora, into isoflavones, flavanones, isoflavanones, isoflavans, and pterocarpan, are shown in Table 1. Each compound (200substrate. screened for o-diphenolase inhibitory activity of mushroom tyrosinase using L-DOPA as the μM) was screened for o-diphenolase inhibitory activity of mushroom tyrosinase using L -DOPA as the 50% of A total of 11 active inhibitors from the preliminary screening were then determined for their substrate. A total of 11 active inhibitors from the preliminary screening were then determined for their inhibition (IC50 ) values, and the results are summarized in Table 2. 50% of inhibition (IC50) values, and the results are summarized in Table 2.

Table 1. Chemical structures of tested compounds from the heartwood of Dalbergia parviflora. Table 1. Chemical structures of tested compounds from the heartwood of Dalbergia parviflora.

Isoflavones

No. No. 1 1 2 2 3 3 4 4 55 66 77 88 99 No. 10

IsoflavonesIsoflavonesR5 Formononetin H Formononetin Calycosin H Calycosin Biochanin A OH Biochanin A Genistein OH Genistein Khrinone B OH Khrinone B 3′-O-Methylorobol 30 -O-Methylorobol OH Khrinone C C OH Khrinone Tectorigenin OH Tectorigenin Cajanin OH Cajanin Isoflavanones R5 (3R)-7,3′Dihydroxy-4′H methoxyisoflavanone

R6 R5 R7 R6 H OH H H H OH H H H OH OH H H OH OH H H OH OH H H OH OH H H OH OH H OMe OH OHOMe H OH OMeH R6 R7 H

OH

R2′ R7 H OH H OH H OH H OH OH OH HOH OMe OH HOH OH OMe R2′ H

R3′ R20 H H OH H H H H H H OH OMe H OH OMe H H H OH R3′

0 R4′ R3

OMe H OMe OH OMe H OH H OMe H OH OMe OMe OH OH H OH H R4′

OH

OMe

0 R4R5′

H OMe H OMe H OMe OHH OH OMe OHH H OMe OHH OHH R5′ H

R50 H H H H OH H H H H

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Molecules 2017, 22, x FOR PEER REVIEW Molecules 2017, 22, x FOR PEER REVIEW Molecules 2017, 22, x FOR PEER REVIEW No. 11 11 Molecules 2017, 22, x FOR PEER REVIEW 12 12 11 10 13 13 12 11 14 11 14 13 12 12 15 14 13 15 13 14 15 1614 16 15 15 16 1716 Isoflavanones 17 17 Isoflavanones 16 18 17 18 Isoflavanones 18 No. 17 No. No. 18 Isoflavanones 19 19 No. 18 19 20 19 No. 20 20 19 20 2121 21 20 22 21 22 22 23 21 Isoflavans 22 23 23 Isoflavans No. 22 23 Isoflavans No. 24 No. 23 Isoflavans 24 No. 25 25 24 No. 24 25 24 26 25 26 25 Flavanones 26 Flavanones No. 26 26 No. Flavanones No. Flavanones

Pterocarpan Pterocarpan Pterocarpan

Table 1. Cont. OnogeninIsoflavanonesH H R5 Onogenin H H Dalparvin H H 0 -Dihydroxy-4 0(3R)-7,3 Dalparvin Onogenin H H Dalparvin B H H H methoxy-isoflavanone B Dalparvin H H Onogenin H H H (3S)-Sativanone H H Onogenin (3S)-Sativanone Dalparvin B H H Dalparvin H H H (3R,S)-3′-ODalparvin H H (3R,S)-3′-O(3S)-Sativanone H H Methylviolanone Dalparvin BB H H H H H Dalparvin Methylviolanone (3R,S)-3′-O(3S)-Sativanone H H (3R,S)-Kenusanone H H (3S)-Sativanone OH H H (3R,S)-Kenusanone 0 -O-Methylviolanone GMethylviolanone (3R,S)-3′-OOH H (3R,S)-3 H H H G (3R,S)-Kenusanone Methylviolanone (3S)-Secundiflorol OH H OH (3R,S)-Kenusanone OH G H (3S)-Secundiflorol G H (3R,S)-Kenusanone H (3S)-Secundiflorol HOH OH H OH H (3S)-Secundiflorol GDalparvin Dalparvin A A OH H OH H OH Dalparvin A OH H H Isoflavans R6 R7 (3S)-Secundiflorol OH H R6 Isoflavans OH R6 R7 Dalparvin A H H Isoflavans (3R)-Vestitol H OH (3R)-Vestitol H OH Isoflavans R6 R7 Dalparvin A OH H H (3R)(+)(3R)-Vestitol H OH (3R)(+)(3R)-Vestitol H OH Mucronulatol Isoflavans R6 R7 H (3R)(+)-Mucronulatol H OH Mucronulatol (3R)(+)(3R,S)-3′-Hydroxy(3R)-Vestitol H OH (3R,S)-30 -Hydroxy-8-methoxy H OH H OH H (3R,S)-3′-HydroxyMucronulatol 8-methoxy (3R)(+)vestitol vestitol H OH H OH 8-methoxy vestitol (3R,S)-3′-HydroxyDuartin H OH Mucronulatol H OH Duartin vestitol Duartin H OH H 8-methoxy (3S)-8(3R,S)-3′-HydroxyH OH H OH (3S)-8Duartin H OH Demethylduartin 8-methoxy vestitol H OH H (3S)-8-Demethylduartin Demethylduartin (3S)-8Flavanones R3 R5 Duartin H OH H OH Flavanones R3 R5 Demethylduartin (2S)-Liquiritigenin H (3S)-8Flavanones HH R3 OH (2S)-Liquiritigenin H H Flavanones R3 R5 Demethylduartin (2S)-Naringenin H OH (2S)-Naringenin OH (2S)-Liquiritigenin H H Flavanones R3 R5 (2S)-Liquiritigenin H H (2S)-Naringenin OH (2S)-Liquiritigenin H H Alpinetin H OMe (2S)-Naringenin H OH Alpinetin H OMe

(2S)-Naringenin

Alpinetin H Pterocarpan R5 Alpinetin Alpinetin H Pterocarpan R5 Pterocarpan R5 No. (6aR,11aR)-3,8Pterocarpan R5 No. Pterocarpan (6aR,11aR)-3,8Dihydroxy-927 H Dihydroxy-9(6aR,11aR)-3,8methoxy 27 H methoxy Dihydroxy-9pterocarpan (6aR,11aR)-3,827 H pterocarpan methoxy Dihydroxy-9(6aR,11aR)-3,8-Dihydroxy27 H 27 pterocarpan methoxy 9-methoxy pterocarpan pterocarpan

H

OMe R6 H OMe R6 R6

OHR6 OH OH OH OHH OH OHH OH OH OHH OH OH OHH OH OH OH OHH OH OHH OHH OH OH OHH OH OHH OH R8 OH R8R7 OH H H R8 OH OH H HOH R8 H H H OMe OH OMe H OMe OMe OH OMe OH OMe OMe OH OH R6 OMe OH R6 H R5 OH H R6 H H R6 H H H H H H

OH

H R7OMe H R7 R7

R6 R5 R7R6 H OH H OH H OH H H

OHH

R7 OMe OMe OMe OMe OH OH OH OMe OMe OMe OH OMe OH OMe OH OMe OMe OH OMe OH OMe OMe OH H H OH OMe H OH OMe OMe OH H OMe OMe OH OMe R2′ OMe R8 R2′ OMe OH OH R2′ OMe H OMe OH R2′ H OMe OH OMe OH OMe OH OMe OMe OH OMe OMe OMe OH OMe OMe OH R7 OMe OMe R7 OH R6 OMe OH R7 OH OH R7 H OH OH OH OH OH H

OH R2′ H OH R2′ R2′

R2 H0

H H

HH OMe

OMe H

H H OMe H OMe H OMe OMe H OMe OMe OH H OMe OMe OH OH OMe OMe OH H OH OH OMe OH H OH OMe H R3′

OH R2 R3′ H0 H

H R3′ H OH OH H R3′ OMe OH H

OH OH OH

OH OH OH OH OMe OH OH OH OH OH OMe R4′ OH OH R4′ OH R7 OH OH R4′ OH OH R4′ OH OH OH H OH H

OH

H R3′ OH H R3′ R3′

R7 R2′ H H H

0 R2 R3′

H OH

OH H

OH OH OH

3 of 11 3 of 11 3 ofR4 110 0 R3 OCH R50 2O OCH O 3 of 211 OMe OH OMe OCH 2OOH OH OMe H OMe H H OMe OH OCH 2OH OMe H OCH2 O OMe H OMe OMe OH H OH OMe H OMe H OMe H H OMe OMeH OMe OMe OMeH H H OMe H OMe H OMe OMe OMe OMeH H H OMe OMe H H OH OMe H OMe OH OMe OMeH H H OMe OH OMe OMe H OH H OMe OH R4′ R5′ 0 OMe H R50 R3 R40OH R4′ R5′ OMe OMe H OMe H R4′ R5′ OH H HOMe OMe OMe H OMe H H R4′ OMe R5′ OH OMe H OMe H OMe H OMe H OH OMe H OMe H OMe H OMe H OMe H OH OMe OMeH H OMe H OMe H OMe H OMe OMeH H OH R5′ R6′ OMe H OMe H R5′ R6′ 0 H R4OMe R50HH R60 H H R5′ R6′ H H H H R5′ R6′ OHH H H H H H H H H H H H

OH

H HR4′ H R4′

H

H HR5′ H R5′

R4′

R5′

OMe OMe OMe

H H H

H

H

0 R3R4′ R40R5′ R50

OMe OMeH OH

H

Table 2. Mushroom tyrosinase inhibitory activities of flavonoids extracted from Dalbergia parviflora. Table 2. Mushroom tyrosinase inhibitory activities of flavonoids extracted from Dalbergia parviflora. Pterocarpan Table 2. Mushroom tyrosinase inhibitory activities of flavonoidsMushroom extracted from DalbergiaInhibition parviflora. Tyrosinase Mushroom Tyrosinase Inhibition No. Compound Inhibition Table 2. Mushroom tyrosinase inhibitory activities of flavonoids% extracted from Dalbergia parviflora. Mushroom TyrosinaseICInhibition 50 (µM) No. Compound % (atInhibition 200 µM) with a hydroxyl From the nine isoflavones (compounds 1–9), only two compounds IC50 (µM) group at the No. Compound % Inhibition (at 200 µM) Mushroom Tyrosinase Inhibition (control) of Oxyresveratrol 98.4 ±o-diphenolase 1.1 0.19 0.1 ICinhibitory 50 ± (µM) C-20 position ring B—khrinone B (5) and cajanin (9)—exhibited potential, (at 200± µM) (control) Oxyresveratrol 98.4 1.1 0.19±±4.6 0.1 No. Compound % Inhibition (control) Kojic acid 93.4 ± 1.7 16.8 IC 50 (µM) with IC(control) values of 54.0 ± 6.0 and 67.9 ± 6.2 µM, respectively. The other five isoflavones—namely Oxyresveratrol 98.4 ± µM) 1.1 0.19 0.1 Kojic acid 93.4 1.7 16.8±±5.0 4.6 5027 (at 200 (6aR,11aR)-3,8-Dihydroxy-9-methoxy pterocarpan 84.6 ± 0.6 16.7 (control) Kojic acid 93.4 1.7 16.8 4.6 27 (6aR,11aR)-3,8-Dihydroxy-9-methoxy pterocarpan 84.6 0.6 16.7 5.0 (control) Oxyresveratrol 98.4±±±2.2 1.1 0.19±±±6.0 0.1C (7)—did not formononetin (1), calycosin (2), biochanin A (3) 30 -O-methylorobol (6), and khrinone 5 Khrinone B 72.7 54.0 27 (6aR,11aR)-3,8-Dihydroxy-9-methoxy pterocarpan 84.6 ±± 0.6 16.7 ±± 5.0 5 Khrinone B 72.7 2.2 54.0 6.0 (control) Kojic acid 93.4 1.7 16.8 4.6 calycosin has 21 (3RS)-3′-Hydroxy-8-methoxy 64.1to ± 1.3 ± 5.8 show inhibitory activity on mushroomvestitol tyrosinase. In contrast a previous67.8 report, 5 Khrinone B 72.7 ±± 2.2 54.0 ±± 6.0 21 (3RS)-3′-Hydroxy-8-methoxy vestitol 64.1 1.3 67.8 5.8 27 (6aR,11aR)-3,8-Dihydroxy-9-methoxy pterocarpan 84.6 0.6 16.7 5.0 9 to be Cajanin 65.0 ± 1.6 with an 67.9 ± 6.2 been shown a monophenolase inhibitor IC of 38.4 µM. 50±± value 21 (3RS)-3′-Hydroxy-8-methoxy vestitol of mushroom tyrosinase 64.1 1.3 67.8 5.8 Cajanin 65.0 1.6 67.9 6.2 Khrinone B 72.7±±±0.4 2.2 54.0 6.0 1095 (3R)-7,3′-Dihydroxy-4′-methoxy-isoflavanone 52.1 176.7 ± 16.3 9 calycosin Cajanin 65.0 ±± 1.6 67.9 ±± 6.2 In addition, reduced melanin biosynthesis in murine melan-a melanocytes [13]. Most of 10 (3R)-7,3′-Dihydroxy-4′-methoxy-isoflavanone 52.1 0.4 176.7 16.3 21 (3RS)-3′-Hydroxy-8-methoxy vestitol 64.1 1.3 67.8 5.8 24 (2S)-Liquiritigenin 52.1 ± 1.4 178.1 ± 14.0 10 (3R)-7,3′-Dihydroxy-4′-methoxy-isoflavanone 52.1 ±± 0.4 176.7 ±± 16.3 24 (2S)-Liquiritigenin 1.4 178.1 14.0 9 Cajanin 65.0 1.6 67.9 6.2 the isoflavanones, isoflavans, and flavanones showed poor or no inhibitory activity on tyrosinase. 20 (3R)(+)-Mucronulatol 48.3 ± 1.6 228.9 ± 22.2 24 (2S)-Liquiritigenin 52.1 1.4 178.1 14.0 20 (3R)(+)-Mucronulatol 48.3 1.6 228.9 22.2 10 (3R)-7,3′-Dihydroxy-4′-methoxy-isoflavanone 52.1±±±3.7 0.4 176.7±±±54.5 16.3 17 (3S)-Secundiflorol Hvestitol (21), an isoflavan with 44.0 278.1 (3R,S)-30 -hydroxy-8-methyoxy hydroxyl groups at the R20 and R30 20 (3R)(+)-Mucronulatol 48.3 ±± 1.6 228.9 ±± 22.2 17 (3S)-Secundiflorol H 44.0 3.7 278.1 54.5 24 (2S)-Liquiritigenin 52.1 1.4 178.1 14.0 26 Alpinetin 36.9 ± 0.5 450.0 ± 48.5 positions of B, showed inhibition with an IC50 value of 67.8 ± 5.8 µM. Pterocarpan (27) was the best 17 (3S)-Secundiflorol H 44.0 ± 3.7 278.1 54.5 26 Alpinetin 36.9 0.5 450.0 48.5 20 ring(3R)-Vestitol (3R)(+)-Mucronulatol 48.3 1.6 228.9±±±60.9 22.2 19 35.6 ±±2.2 473.0 26 Alpinetin 36.9 ± 0.5 450.0 ± 48.5 19 (3R)-Vestitol 35.6 2.2 473.0 60.9 mushroom tyrosinase inhibitor among the 27 flavonoids tested, with an IC value equal 17 (3S)-Secundiflorol H 44.0±±0.5 3.7 278.1±±43.6 54.5 to kojic acid 50 906.1 12 Dalparvin 31.6 19 (3R)-Vestitol 35.6 ± 2.2 473.0 ± 60.9 12 Dalparvin 31.6 0.5 906.1 43.6 26 Alpinetin 36.9 ± 450.0 ± (16.7 ± 5.06 µM and 16.8 ± 4.8 µM, respectively). Moreover, the positive exhibited 3′-O-Methylorobol 14.3 ± 1.1 control, oxyresveratol, N.D.48.5 12 Dalparvin 31.6 ±± 0.5 906.1 ±± 43.6 6 3′-O-Methylorobol 14.3 1.1 N.D. 19 (3R)-Vestitol 35.6 2.2 473.0 60.9 3 Biochanin Aeffect on mushroom tyrosinase with 9.2 ±IC 0.450 of 0.19 ± N.D. the strongest inhibitory an 0.06 µM. The crude 63 3′-O-Methylorobol 14.3 1.1 N.D. Biochanin 9.2 ±±±0.4 Dalparvin A 0.5 906.1 ± 43.6 112 Formononetin 10%31.6 (at 300 μM) N.D. extract of D. exhibited tyrosinase inhibitory activity with IC50 of 2.6 ± 0.4 µg/mL. 3 parviflora Biochanin A 9.2 ±300 0.4 N.D. Formononetin 10% (at300 μM) 3′-O-Methylorobol 14.3 ±an 1.1 N.D. 716 Khrinone C 2% (at μM) N.D. 173 Formononetin 10% (at300 300 μM)interaction N.D. Khrinone C 2% (at μM) The inhibition of enzymes by certain flavonoids may be due to the Biochanin A 9.2 ± 0.4 N.D. 2 Calycosin 0% (at 300 μM) N.D.of the flavonoids 721 Khrinone C 2% (at 300 μM) N.D. Calycosin 0%[14] Formononetin 10% (at300 300 μM) N.D. with free11 radicals generated at the active sites of the enzymes orμM) with copper ion in the catalytic Onogenin 0% (at N.D. 27 Calycosin 0% (at 300 μM) N.D. 11 Onogenin Khrinone C 2% (at 300 μM) N.D. 15 (3R,S)-3′-O-Methylviolanone 0% (at 300 μM) N.D. domains 11 of the Onogenin enzymes [7]. The numbers and positions 0% of (at the300hydroxyl groups attached to the μM) N.D. 15 (3R,S)-3′-O-Methylviolanone 2 Calycosin B 0%(at (at500 300μM) μM) N.D. 13 Dalparvin 4% N.D. flavonoids skeletons are the key to the inhibitory activity of mushroom tyrosinase. Chang et al. 15 (3R,S)-3′-O-Methylviolanone 0% (at 300 μM) N.D. 13 Dalparvin B 4% 500 11 Onogenin 0% (at 300 μM) N.D. 0 -trihydroxyisoflavone, 0 -dihydroxyisoflavone), 13 B 4% μM) N.D. 15 6,7,4Dalparvin (3R,S)-3′-O-Methylviolanone 0% (at (at 500 300 μM) N.D. found that glycitein (6-methoxy-7,4 daidzein 0 0 13 Dalparvin B 4% (at 500 μM) N.D. (7,4 -dihydroxyisoflavone), and genistein (5,7,4 -trihydroxyisoflavone) inhibited the monophenolase

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activity of mushroom tyrosinase with IC50 values of 9.2, 237, 264, and 822 µM, respectively. The results suggest that the hydroxyl groups at the C6 and C7 positions of ring A of the isoflavone skeleton might play an important role in the expression of monophenolase inhibitory activity [15]. Chang et al. also found that 7,8,40 -trihydroxyisoflavone and 5,7,8,40 -tetrahydroxyisoflavone irreversibly inhibit the monophenolase and diphenolase activities of tyrosinase. The study suggested that the presence of hydroxyl groups at the C7 and C8 positions of the isoflavone skeleton may also cause this irreversible inhibition [16]. Table 2. Mushroom tyrosinase inhibitory activities of flavonoids extracted from Dalbergia parviflora. No.

Compound

(control) (control) 27 5 21 9 10 24 20 17 26 19 12 6 3 1 7 2 11 15 13 14 16 18 22 4 8 23 25

Oxyresveratrol Kojic acid (6aR,11aR)-3,8-Dihydroxy-9-methoxy pterocarpan Khrinone B (3RS)-30 -Hydroxy-8-methoxy vestitol Cajanin (3R)-7,30 -Dihydroxy-40 -methoxy-isoflavanone (2S)-Liquiritigenin (3R)(+)-Mucronulatol (3S)-Secundiflorol H Alpinetin (3R)-Vestitol Dalparvin 30 -O-Methylorobol Biochanin A Formononetin Khrinone C Calycosin Onogenin (3R,S)-30 -O-Methylviolanone Dalparvin B (3S)-Sativanone (3R,S)-Kenusanone G Dalparvin A Duartin Genistein Tectorigenin (3S)-8-Demethylduartin (2S)-Naringenin

Mushroom Tyrosinase Inhibition % Inhibition (at 200 µM)

IC50 (µM)

98.4 ± 1.1 93.4 ± 1.7 84.6 ± 0.6 72.7 ± 2.2 64.1 ± 1.3 65.0 ± 1.6 52.1 ± 0.4 52.1 ± 1.4 48.3 ± 1.6 44.0 ± 3.7 36.9 ± 0.5 35.6 ± 2.2 31.6 ± 0.5 14.3 ± 1.1 9.2 ± 0.4 10% (at 300 µM) 2% (at 300 µM) 0% (at 300 µM) 0% (at 300 µM) 0% (at 300 µM) 4% (at 500 µM) 13% (at 500 µM) 0% (at 500 µM) 0% (at 500 µM) 0% (at 500 µM) S* S* S* S*

0.19 ± 0.1 16.8 ± 4.6 16.7 ± 5.0 54.0 ± 6.0 67.8 ± 5.8 67.9 ± 6.2 176.7 ± 16.3 178.1 ± 14.0 228.9 ± 22.2 278.1 ± 54.5 450.0 ± 48.5 473.0 ± 60.9 906.1 ± 43.6 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

N.D.: not determined; S*: These flavonoids can react with mushroom tyrosinase to form a colored product that interferes with the spectrophotometric measurement.

In the series of flavonoids in this study, only (3S)-8-demethylduartin (23) contained 7,8-dihydroxy groups. However, the inhibitory activities of khrinone B (5) and cajanin (9) suggested that not only the 6,7- or 7,8-dihydroxy groups of ring A, but also the hydroxyl group at the C20 position of ring B of isoflavone or isoflavanone skeletons, might play important roles in the expression of o-diphenolase inhibitory activity. The inhibitory activities of (3RS)-30 -hydroxy-8-methoxy vestitol (21) and (6aR,11aR)-3,8-dihydroxy-9-methoxy pterocarpan (27) supported the importance of both the hydroxyl group at the C30 position on ring B of isoflavans and pterocarpan for o-diphenolase inhibitory activity. In this study, the 7,8-dihydroxy groups are only present in (3S)-8-demethylduartin (23), which could be a potent tyrosinase inhibitor. However, after pre-incubation of (3S)-8-demethylduartin (23) with the enzyme in the absence of the substrate (L-DOPA), an orange metabolite with an optical density at 475 nm was observed. This observation suggests that (3S)-8-demethylduartin (23) can act as a chromogenic substrate analog for mushroom tyrosinase. The other flavonoids that were found to be chromogenic substrate analogs include genistein (4), tectorigenin (8), and (2S)-naringenin (25). These results are comparable to a previous report in which resveratrol, a phenylpropanoid,

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was oxidized by mushroom tyrosinase and converted to a newly oxidized compound which was observed at 475 nm and became a tyrosinase inhibitor [17,18]. Moreover, Gasowska-Bajger and Wojtasek demonstrated that the oxidation of quercetin, kaempferol, morin, catechin, and naringenin by mushroom tyrosinase strongly influenced the measurement of the absorbance at 475 nm during oxidation of L-tyrosine or L-DOPA, generating false inhibition or activation effects [19]. 2.2. Inhibitory Effect of Flavonoids on the o-Diphenolase Activity of Murine Tyrosinase The inhibition of murine tyrosinase was studied using the flavonoids at a concentration of 200 µM. The results revealed that among the 27 flavonoids, only (6aR,11aR)-3,8-dihydroxy-9-methoxy pterocarpan (27) showed a weak inhibitory effect on the o-diphenolase activity of murine tyrosinase with an inhibition of 29.2 ± 2.9% (Table 3). The crude extract and the other flavonoids did not show any inhibition (data not shown), whereas 200 µM of kojic acid and oxyresveratrol, the positive controls, showed strong inhibitory effects of 73.8 ± 1.0% (IC50 = 39.9 µM) and 99.4 ± 0.9% (IC50 = 0.88 ± 0.16 µM), respectively. Table 3. Murine tyrosinase inhibitory activity and melanogenesis of flavonoids from Dalbergia parviflora.

No. 1 2 3 4 5 6 7 8 9 No. 10 11 12 13 14 15 16 17 18 No. 19 20 21 22 23 No. 24 25 26 No. 27

Isoflavones Formononetin Calycosin Biochanin A Genistein Khrinone B 30 -O-Methylorobol Khrinone C Tectorigenin Cajanin

Murine Tyrosinase Inhibition, % (at 200 µM) 9.9 ± 3.1 10.1 ± 3.0 10.2 ± 2.9 9.2 ± 1.5

% Melanin Content

% Cell Viability

% Melanin Content

% Cell Viability

133.8 ± 2.7 142.8 ± 8.2 165.2 ± 5.9 165.1 ± 1.6 84.1 ± 5.3 170.1 ± 10.5 107.6 ± 0.8 127.6 ± 2.8 65.8 ± 7.5

96.0 ± 3.3 99.1 ± 9.2 103.4 ± 4.5 128.2 ± 3.5 95.1 ± 5.4 106.6 ± 6.2 93.3 ± 2.0 129.7 ± 3.4 101.5 ± 6.6

155.3 ± 15.4 196.6 ± 6.4 168.0 ± 5.1 191.7 ± 13.9 34.7 ± 12.3 215.7 ± 15.7 154.0 ± 9.9 152.1 ± 4.7 61.6 ± 8.9

92.8 ± 9.4 101.3 ± 1.9 102.2 ± 4.8 93.8 ± 5.7 62.3 ± 15.1 101.5 ± 2.7 93.4 ± 2.0 119.3 ± 1.3 107.2 ± 3.3

-

95.1 ± 3.0

94.9 ± 3.9

95.2 ± 2.7

96.5 ± 3.9

9.8 ± 2.8 9.5 ± 1.6 -

133.5 ± 4.6 133.8 ± 9.7 102.0 ± 2.7 124.2 ± 4.3 143.3 ± 8.4 132.6 ± 5.1 117.6 ± 17.7 132.0 ± 4.4

100.0 ± 5.0 117.1 ± 8.9 82.3 ± 5.4 106.7 ± 10.6 74.2 ± 7.8 124.0 ± 1.7 109.4 ± 6.6 105.4 ± 4.8

169.8 ± 15.4 199.6 ± 12.1 99.1 ± 2.3 127.6 ± 6.0 157.2 ± 11.5 158.5 ± 13.5 146.4 ± 6.1 184.2 ± 10.6

92.0 ± 2.0 99.7 ± 6.7 74.1 ± 6.4 64.3 ± 23.7 72.9 ± 6.2 102.2 ± 2.8 102.7 ± 4.1 100.4 ± 7.1

12.4 ± 3.3 12.8 ± 2.1

186.1 ± 1.6 178.6 ± 15.9

142.1 ± 3.0 103.6 ± 6.9

232.0 ± 9.3 263.7± 8.2

119.7 ± 4.1 102.5 ± 2.7

11.4 ± 2.5

97.0 ± 6.3

102.9 ± 1.0

36.3 ± 0.1

62.4 ± 3.2

-

301.1 ± 2.3 87.8 ± 2.0

139.9 ± 7.5 93.5 ± 3.0

326.8 ± 2.1 72.7 ± 4.3

94.4 ± 2.8 98.3 ± 5.4

13.2 ± 2.7 -

102.9 ± 5.5 132.5 ± 14.5 89.7 ± 0.4

98.9 ± 10.7 94.3 ± 2.0 77.0 ± 12.7

124.5 ± 2.0 152.1 ± 3.8 92.3 ± 1.6

100.0 ± 3.5 98.1 ± 0.5 77.1 ± 10.2

29.2 ± 2.9

41.9 ± 0.4

105.7 ± 4.9

53.5 ± 0.4

14.5 ± 0.9

73.8 ± 1.0 99.4 ± 1.0

68.9 ± 5.2 24.3 ± 1.1

98.9 ± 4.7 117.2 ± 2.2

74.2 ± 5.8 28.4 ± 1.7

99.6 ± 2.3 118.1 ± 5.1

15 µM

30 µM

Isoflavanones (3R)-7,30 -Dihydroxy-40 methoxy-isoflavanone Onogenin Dalparvin Dalparvin B (3S)-Sativanone (3R,S)-30 -O-Methylviolanone (3R,S)-Kenusanone G (3S)-Secundiflorol H Dalparvin A Isoflavans (3R)-Vestitol (3R)(+)-Mucronulatol (3RS)-30 -Hydroxy-8-methoxy Vestitol Duartin (3S)-8-Demethylduartin Flavanones (2S)-Liquiritigenin (2S)-Naringenin Alpinetin Pterocarpan (6aR,11aR)-3,8-Dihydroxy-9-methoxy pterocarpan Others

Control Kojic acid Control Oxyresveratrol

-: no inhibition at 200 µM.

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Notably, the flavonoids exhibited stronger effects on mushroom tyrosinase than murine tyrosinase activity. In fact, almostchampignon all studies conducted tyrosinase inhibition have Although used the commercially commercially available mushroomon (Agaricus bisporus) tyrosinase. these results available champignon mushroom (Agaricuscan bisporus) tyrosinase. Although these results may highlight may highlight that mushroom tyrosinase be used for anti-tyrosinase activity screening, it does that mushroom tyrosinase can be used for anti-tyrosinase activity screening, it does not appear to be not appear to be sufficiently accurate to measure the potential of the active substance as a whitening sufficiently accurate to measure the potential of the active substance as a whitening agent. A more agent. A more appropriate method would be to use the mammalian tyrosinase inhibition assay and appropriate method would be tofrom use the tyrosinase inhibition assay tyrosinase and melanogenesis melanogenesis inhibition results the mammalian cell-based assay. However, mushroom is still a inhibition results from the cell-based assay. However, mushroom tyrosinase is still a suitable screening suitable screening tool to measure the anti-browning activity of plant-derived foods in the food tool to measure the anti-browning activity of plant-derived foods in the food industry. industry. 2.3.Effects Effectsofofthe theFlavonoids Flavonoidson onCell CellViability Viabilityand andMelanogenesis MelanogenesisofofB16-F10 B16-F10Melanoma MelanomaCells Cells 2.3. MurineB16-F10 B16-F10 melanoma melanoma cells cells were the 2727 flavonoids on Murine were used usedas asaamodel modeltotoexamine examinethe theeffects effectsofof the flavonoids cellcell viability and and melanogenesis (Table(Table 2). The2). melanin content content was measured at 405 nm,ata wavelength on viability melanogenesis The melanin was measured 405 nm, a at which both pheomelanin and eumelanin absorb light [20]. The reason for usingfor phenol wavelength at which both pheomelanin and eumelanin absorb light [20]. The reason using red-free phenol supplemented DMEM was that as cells grow, they produce lactic acid during cellular metabolism, red-free supplemented DMEM was that as cells grow, they produce lactic acid during cellular which causeswhich the pH of thethe medium decrease. This decrease changes the phenol red DMEM medium metabolism, causes pH oftothe medium to decrease. This decrease changes the phenol red to yellow, which interferes with the measurement of the brown melanin production recorded at 405 nm. DMEM medium to yellow, which interferes with the measurement of the brown melanin production The results dark-brown cell are showncell in Figure which led to measurement of cell recorded at of 405the nm. The results ofculture the dark-brown culture1,are shown in the Figure 1, which led to viability and total melanin content (Table 3). the measurement of cell viability and total melanin content (Table 3).

Figure assay—the tested compound was added to atofinal concentration of 30of Figure1.1.Total Totalmelanin melanincontent content assay—the tested compound was added a final concentration μM of B16-F10 melanoma grown cells. The compounds genistein (4), duartin 30 µM of B16-F10 melanoma grown cells. The compounds genistein (4), duartin(22), (22),and and(2S)-naringenin (2S)-naringenin (25) melanin content. content. The Theinhibitory inhibitoryeffect effectofofmelanogenesis melanogenesis was found in cajanin (9) (25)increased increased the the melanin was found in cajanin (9) and and (6aR,11aR)-3,8-dihydroxy-9-methoxy pterocarpan Oxyresveratrol wasasused as a (6aR,11aR)-3,8-dihydroxy-9-methoxy pterocarpan (27). (27). Oxyresveratrol (Oxy) (Oxy) was used a positive positive (C) is the negative control which 0.5% dimethyl was added. control. control. (C) is the negative control to whichto0.5% dimethyl sulfoxidesulfoxide (DMSO) (DMSO) was added.

Compounds cytotoxicity against melanoma cells at aatconcentration of 15 Compounds13, 13,15, 15,and and2626exhibited exhibited cytotoxicity against melanoma cells a concentration of μM with the cell viability decreasing to approximately 70–80% of the control. Compounds 5, 14, 15 µM with the cell viability decreasing to approximately 70–80% of the control. Compounds 5, 14,and and 21 at aa concentration concentrationofof30 30µM μMwith with a cell viability of 60%. highest cytotoxicity 21showed showed toxicity toxicity at a cell viability of 60%. TheThe highest cytotoxicity was was found in compound 27, with a cell viability of 14% at 30 μM, but no toxicity was exhibited atµM. 15 found in compound 27, with a cell viability of 14% at 30 µM, but no toxicity was exhibited at 15 μM. Compounds 8, 16, 19, and 22 increased the quantity of melanoma cells with a cell viability of Compounds 8, 16, 19, and 22 increased the quantity of melanoma cells with a cell viability of 120–140% 120–140% with at thea concentration control at a of concentration of 15 μM.effect Theofinhibitory effectwas of compared compared with the control 15 µM. The inhibitory melanogenesis melanogenesis was found in compounds 9 and 27. Although compound 27 exhibited 60% inhibition found in compounds 9 and 27. Although compound 27 exhibited 60% inhibition of melanogenesis at of at 15 μM affecting the cellcytotoxicity viability, it showed at 30compound μM (Table9 15melanogenesis µM without affecting thewithout cell viability, it showed at 30 µMcytotoxicity (Table 3). Only 3). Only compound 9 significantly the cellular melanin content without affecting the cells significantly decreased the cellulardecreased melanin content without affecting the cells at both concentrations. at both concentrations. From the 27 flavonoids tested, most of them increased melanin production, especially Duartin From the 27 flavonoids tested,increase most of in them increased melanin especially Duartin1 (22), which exhibited a three-fold melanin content with production, respect to the control (Figure (22), which exhibited a three-fold increase in melanin content with respect to the control (Figure 1 and Table 3). Treatment of B16-F10 cells with the D. parviflora crude extract significantly promoted and Table 3). Treatment of B16-F10 cells with the D. parviflora crude extract significantly promoted melanin formation in a dose-dependent manner. At the concentrations of 1.25, 2.5, 5.0, and 10.0 µg/mL melanin formation in a dose-dependent manner. At the concentrations of 1.25, 2.5, 5.0, and 10.0 of the crude extract, the melanin content increased by 158, 173, 226, and 288%, respectively, without μg/mL of the crude extract, the melanin content increased by 158, 173, 226, and 288%, respectively, without exhibiting toxicity (Figure 2). However, at the concentrations of 20, 30, and 40 μg/mL, the cell viability decreased to 85, 80, and 75%, respectively.

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exhibiting toxicity (Figure 2). However, at the concentrations of 20, 30, and 40 µg/mL, the cell viability decreased to 85, 80, and 75%, respectively.

FigureFigure 2. D. 2. parviflora crude extract induced melanogenesis in in B16-F10 melanoma cells. D. parviflora crude extract induced melanogenesis B16-F10 melanoma cells.The Thecells cells were were treated with the crude extract for 72 h. Data represent means ± SDs. The melanin content was treated with the crude extract for 72 h. Data represent means ± SDs. The melanin content was significantly different between the control and treatment groups (p < 0.05). significantly different between the control and treatment groups (p < 0.05).

A previous reportreport indicated that quercetin and naringenin stimulate melanogenesis in cultured A previous indicated that quercetin and naringenin stimulate melanogenesis in cultured murine B16-F10 melanoma cells. Kubo et al. suggested that the improvement of melanin production murine B16-F10 melanoma cells. Kubo et al. suggested that the improvement of melanin production by quercetin may be due, part, melanocytotoxicity [21], whereas Nagata et al. et suggested that that by quercetin may be in due, in to part, to melanocytotoxicity [21], whereas Nagata al. suggested quercetin stimulates melanogenesis by increasing tyrosinase activity and decreasing otherother factors, quercetin stimulates melanogenesis by increasing tyrosinase activity and decreasing factors, such such as melanogenic inhibitors [22].[22]. Ohguchi et al. [23] and Bouzaiene as melanogenic inhibitors Ohguchi et al. [23] and Bouzaieneetetal.al.[24] [24]also alsoreported reported that that naringenin of of melanogenic enzymes in B16-F10. Takekoshi et al.et al. naringenin increases increasesthe theexpression expressionlevel level melanogenic enzymes in B16-F10. Takekoshi reported that flavonoids, including quercetin, kaempferol, rhamnetin, fisetin, apigenin, luteolin, reported that flavonoids, including quercetin, kaempferol, rhamnetin, fisetin, apigenin, luteolin, chrysin, and genestein, showed melanogenesis-promoting actions on human melanoma cells (MMV chrysin, and genestein, showed melanogenesis-promoting actions on human melanoma cells (MMV II), which promoted melanogenesis by flavonoids that was on increased intracellular II), which promoted melanogenesis by flavonoids that dependent was dependent on increased intracellular tyrosinase activity. Moreover, the relationship between the structure and melanogenesis-promoting tyrosinase activity. Moreover, the relationship between the structure and melanogenesis-promoting actions of theofflavonoids suggested that the groupgroup bound to thetophenyl groupgroup playsplays an an actions the flavonoids suggested thathydroxyl the hydroxyl bound the phenyl important role inrole theinstimulation of melanogenesis [25]. In this the comparison of theofchemical important the stimulation of melanogenesis [25]. Instudy, this study, the comparison the chemical structures between duartin (22) and (23) revealed that the structures between duartin (22) (3S)-8-demethylduartin and (3S)-8-demethylduartin (23) revealed that7,8-dihydroxy the 7,8-dihydroxy structure in theinisoflavans was associated with melanogenesis inhibition. The R8-OMe substitution structure the isoflavans was associated with melanogenesis inhibition. The R8-OMe substitution in duartin (22) had strong effect effect on melanogenesis stimulation. The comparison of chemical in duartin (22) ahad a strong on melanogenesis stimulation. The comparison of chemical 0 structures between (3RS)-3 -hydroxy-8-methoxy vestitol (21) and (22) also that the structures between (3RS)-3′-hydroxy-8-methoxy vestitol (21)duartin and duartin (22)revealed also revealed that the 20 ,30 -dihydroxy structure was associated with with an inhibition of mushroom tyrosinase. Duartin (22), (22), 2′,3′-dihydroxy structure was associated an inhibition of mushroom tyrosinase. Duartin 0 -methoxy associated with 2with structure, exhibited the strongest melanogenesis stimulation and loss associated 2′-methoxy structure, exhibited the strongest melanogenesis stimulation andof loss of mushroom tyrosinase inhibitory activity. mushroom tyrosinase inhibitory activity. All tyrosinases have have a binuclear type type threethree copper center within their their activeactive site in common. All tyrosinases a binuclear copper center within site in common. However, no common tyrosinase protein structure has been foundfound to occur across all species [26–29]. However, no common tyrosinase protein structure has been to occur across all species [26–29]. ThereThere are significant differences between mushroom tyrosinase and mammalian tyrosinase in termsinofterms are significant differences between mushroom tyrosinase and mammalian tyrosinase structural properties, molecular properties, kinetic kinetic properties, and localization. Tyrosinase from from of structural properties, molecular properties, properties, and localization. Tyrosinase the mushroom A. bisporus hashas been solublecytosolic cytosolic enzyme containing the mushroom A. bisporus beenreported reportedto to be be aasoluble enzyme containing twotwo different different subunits, referred heavy(H) (H)and and light of 43 13.4 13.4 kDa, kDa, subunits, referred to to as asheavy light (L), (L),with withmolecular molecularweights weights of and 43 and respectively. In aqueous solution,solution, the predominant form of the form mushroom is thetyrosinase quaternaryis the respectively. In aqueous the predominant of thetyrosinase mushroom L2H2astructure with a molecular weight of 120 the kDa,active whereas theappears active form L2 H2 quaternary structure with molecular weight of 120 kDa, whereas form to beappears L2 H to L2H withmolecular an apparent molecular weight kDa [30]. In contrast, human tyrosinase is a with be an apparent weight of 69 kDa [30]. ofIn69 contrast, human tyrosinase is a monomeric monomeric membrane-bound a molecular weight 66.7 etkDa Song et al. membrane-bound glycoprotein withglycoprotein a molecularwith weight of 66.7 kDa [29]. ofSong al. [29]. suggested suggested there iscorrelation no significant correlation between the inhibition of mushroom tyrosinase that there is no that significant between the inhibition of mushroom tyrosinase with human with humantyrosinase melanoma tyrosinase and melanogenesis [31].theAinhibition study on ofthe inhibitiontyrosinase of mushroom melanoma and melanogenesis [31]. A study on mushroom tyrosinase and the cellular tyrosinase activities of mulberroside A, oxyresveratrol, resveratrol, and arbutin also suggested that the in vitro inhibition rate of mushroom tyrosinase might not represent the inhibition rate of melanin synthesis for in vivo systems [9]. However, we suggest that, in

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and the cellular tyrosinase activities of mulberroside A, oxyresveratrol, resveratrol, and arbutin also suggested that the in vitro inhibition rate of mushroom tyrosinase might not represent the inhibition rate of melanin synthesis for in vivo systems [9]. However, we suggest that, in flavonoids, anti-tyrosinase activity against mushroom would only appear in parallel with mammalian tyrosinase activity and melanogenesis inhibition when the enzyme inhibition activity in mushroom tyrosinase is greater than 60% at a concentration of 200 µM or an IC50 lower than 50 µM. In other words, most flavonoids are likely to stimulate melanogenesis despite inhibiting mushroom tyrosinase activity. 3. Materials and Methods 3.1. Chemicals 3,4-Dihydroxy-L-phenylalanine (L-DOPA), kojic acid, oxyresveratrol, and mushroom tyrosinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 27 flavonoids from D. parviflora were obtained from Kaoru Umehara [10,11]. 3.2. Mushroom Tyrosinase Inhibitory Assay The test samples, L-DOPA, and tyrosinase enzyme solutions were dissolved in 50% (v/v) dimethyl sulfoxide (DMSO) and 20 mM phosphate buffer solution (PBS) of pH 6.8, respectively. The o-diphenolase activity of mushroom tyrosinase was performed in 96-well plates using a modified method of that performed by Likhitwitayawuid and Sritularak [32]. The reaction mixture consisted of 140 µL of 20 mM phosphate buffer at pH 6.8, 20 µL of test sample solution, and 20 µL of mushroom tyrosinase (100 unit/mL, E.C. 1.14.18.1, Sigma). The mixture was pre-incubated at 25 ◦ C for 10 min. Subsequently, 20 µL of 2.5 mM L-DOPA was added, and the mixture was incubated for 20 min at 25 ◦ C. During the reaction, L-DOPA was converted to dopachrome, which resulted in a change in color from colorless to orange. This change was measured through absorbance at 475 nm. Instead of the sample solution, 50% DMSO was used as the control. The reaction mixture without enzyme served as a blank. The inhibitory activity of the sample was expressed as the concentration that inhibits 50% of enzyme activity (IC50 ). 3.3. Cell Cultures Mus musculus (mouse) melanoma cell line, B16-F10 (ATCC® Number: CRL-6475™, Manassas, VA, USA), was cultured in complete Dulbecco0 s Modified Eagle0 s Medium (DMEM, phenol red-free medium) containing 10% fetal bovine serum, 25 mM glucose, 4 mM L-glutamine, 1 mM sodium pyruvate, 10 µg/mL penicillin, and 10 µg/mL streptomycin, at 37 ◦ C, with 5% carbon dioxide (CO2 ) in a humidified atmosphere. 3.4. Murine Tyrosinase Inhibitory Assay B16-F10 melanoma cells were grown to 100% confluence on 10 cm dishes. The trypsinized cells were washed with cold phosphate buffer (20 mM, pH 6.8) and then disrupted in 1 mL of the buffer containing 1% Triton X-100. The cells were lysed by vortex and incubated at 4 ◦ C for 1 h to solubilize the tyrosinase enzyme. The solution was centrifuged at 13,000× g for 15 min at 4 ◦ C to remove the cell debris. The supernatant was dialyzed against 20 mM sodium phosphate buffer (pH 6.8) to remove the Triton X-100. The dialyzed solution was used as a source of crude murine tyrosinase enzyme. The solution contained 1 mg/mL protein, which was determined using a protein assay kit, based on the Bradford method (Bio-Rad Protein assay, Bio-Rad, Hercules, CA, US). The inhibitory activity was determined as described above for mushroom tyrosinase, but the incubation time was optimized at 2 h and 37 ◦ C.

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3.5. Cell Viability and Total Melanin Content Assay Cell viability and melanin content assay were performed in quadruplicate in at least three independent experiments. Cell viability was taken as the reducing power of the living cells that reduce resazurin-based solution (PrestoBlue™, Invitrogen, Carlsbad, CA, USA) to resorufin, which results in a bright red color and is highly fluorescent. Briefly, B16-F10 cells were cultured in a 96-well plate at a concentration of 10,000 cells/0.1 mL/well. After incubating for 24 h, equal volumes of fresh medium containing various concentrations of the test samples were added to the cells and were incubated for 72 h. Cells were then washed with phosphate buffer, and then 0.1 mL of PrestoBlue™ solution in RMPI 1640 serum free media (1:10) was added to each well. After an hour, the fluorescence was quantified using spectrofluorophotometry (excitation 535 nm, emission 610 nm). The total melanin content, produced both extracellularly and intracellularly, was determined as follows: B16-F10 cells were seeded onto a 24-well plate at a concentration of 50,000 cells/0.5 mL/well and were incubated for 24 h, followed by treatment with either the test compounds or the control (0.5% DMSO), and then incubated for 72 h. For extraction, the total produced melanin and 0.5 mL of 2 N NaOH containing 20% DMSO were added directly to the cell culture medium, mixed by pipetting and then incubated at room temperature for 1 h. The lysed cell extract was centrifuged at 13,000× g for 5 min. Finally, the supernatant was transferred to a 96-well plate and the relative melanin content was detected at 405 nm using a micro plate reader. Kojic acid and oxyresveratrol were used as positive controls. 4. Conclusions In conclusion, the results from the current study suggest that the enzyme-based assay using murine tyrosinase from B16-F10 melanoma cells is more appropriate than the mushroom tyrosinase for screening of natural compounds with potential whitening effects. The B16-F10 cells are also good for a cell-based assay for evaluating the direct effect of test compounds on melanin synthesis. Among all flavonoids from Dalbergia parviflora used in this test, (6aR,11aR)-3,8-dihydroxy-9-methoxy pterocarpan (27) and cajanin (9) showed clear inhibitory activities on both methods. However, some flavonoids, particularly duartin (22), showed induction of cellular melanin synthesis without significant effect on the enzyme activity. The mechanism involved in the observed action is still not clear. Author Contributions: Conceptualization, Worrawat Promden and Wanchai De-Eknamkul; Data curation, Worrawat Promden; Formal analysis, Worrawat Promden and Wittawat Viriyabancha; Investigation, Worrawat Promden; Methodology, Worrawat Promden; Resources, Orawan Monthakantirat, Kaoru Umehara, Hiroshi Noguchi and Wanchai De-Eknamkul; Supervision, Wanchai De-Eknamkul; Validation, Worrawat Promden; Visualization, Worrawat Promden; Writing—original draft, Worrawat Promden; Writing—review & editing, Worrawat Promden and Wanchai De-Eknamkul. Acknowledgments: We would like to acknowledge the financial support from the Center of Excellence on Medical Biotechnology (CEMB), S&T Postgraduate Education and Research Development Office (PERDO), and the Higher Education Research Promotion (HERP) Program of the Office of Higher Education Commission (OHEC), Thailand. Conflicts of Interest: The authors declare no conflict of interest.

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