Bioorganic & Medicinal Chemistry 17 (2009) 35–41

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Tyrosinase inhibitory effects of 1,3-diphenylpropanes from Broussonetia kazinoki Yoon Su Baek a, Young Bae Ryu a, Marcus J. Curtis-Long b, Tae Joung Ha c, Rajesh Rengasamy a, Min Suk Yang a, Ki Hun Park a,* a

Division of Applied Life Science (BK21 program), EB-NCRC, Institute of Agriculture & Life Science, Graduate School of Gyeongsang National University, Jinju 660-701, Republic of Korea b 12 New Road, Nafferton, Driffield, East Yorkshire, YO25 4JP, UK c Yeongnam Agricultural Research Institute, National Institute of Crop Science, RDA, Miryang 627-803, Republic of Korea

a r t i c l e

i n f o

Article history: Received 26 September 2008 Revised 8 November 2008 Accepted 11 November 2008 Available online 18 November 2008 Keywords: Broussonetia kazinoki Diphenolase inhibitor 1,3-Diphenylpropane Kazinol S Kazinol T

a b s t r a c t Six 1,3-diphenylpropanes exhibiting inhibitory activities against both the monophenolase and diphenolase actions of tyrosinase were isolated from the methanol (95%) extract of Broussonetia kazinoki. These compounds, 1–6, were identified as kazinol C (1), D (2), F (3), broussonin C (4), kazinol S (5) and kazinol T (6). The latter two species (5 and 6) emerged to be new 1,3-diphenylpropanes which we fully spectroscopically characterized. The IC50 values of compounds (1, 3–5) for monophenolase inhibition were determined to range between 0.43 and 17.9 lM. Compounds 1 and 3–5 also inhibited diphenolase significantly with IC50 values of 22.8, 1.7, 0.57, and 26.9 lM, respectively. All four active tyrosinase inhibitors (1, 3–5) were competitive inhibitors. Interestigly they all mainfested simple reversible slow-binding inhibition against diphenolase. The most potent inhibitor, compound 4 diplayed the following kinetic parameters k3 = 0.0993 lM1 min1, k4 = 0.0048 min-1, and Kiapp = 0.0485 lM. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Tyrosinase (EC 1.14.18.1), which is also referred to as polyphenoloxidase (PPO), is most widely associated with the production of melanin for the protection of skin from solar radiation. However, this beneficial trait comes hand in hand with some severe vices since the overproduction of melanin result in skin hypigmentation, characterized by age spots, melasma and chloasama, all of which are common human maladies.1–3 It has also been suggested that tyrosinase may contribute to the neurodegeneration associated with Parkinson’s disease.4 Thus the unregulated action of tyrosinase is factor in a number of human diseases etiologies. Tyrosinase inhibition has thus been avidly explored as an avenue for therapies to these diseases. The inhibition of melanin formation by tyrosinase is also applicable to fruit preservation by the alleviation of browning, rendering inhibitors of even broader importance. Tyrosinase itself is an enzyme containing a binuclear copper active site.

Abbreviations: IC50, The inhibitor concentration leading to 50% activity loss; Ki, inhibition constant; Kiapp, apparent Ki; k, rate constant; Vmax, maximum velocity; Km, Michaelis–Menten constant; kobs, apparent first-order rate constant for the transition from vi to vs; vi, initial velocity; vs, steady-state rate; A, absorbance at 475 nm. * Corresponding author. Tel.: +82 55 751 5472; fax: +82 55 757 0178. E-mail address: [email protected] (K.H. Park). 0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2008.11.022

It catalyzes two steps in the conversion of tyrosine to melanin. This process proceeds via 3,4-dihydroxy phenylalanine (DOPA), which is formed by tyrosinase monophenolase activity on tyrosine. The next step is the oxidation of DOPA into DOPA quinine, which is a process deemed diphenolase active.5–7 These quinines spontaneously polymerize to high molecular weight brown pigmented species, known melanin. Tyrosinase inhibitors normally either render the copper within the active site inactive by chelation, obviating the substrate–enzyme interaction, or inhibit oxidation via an electrochemical process.8,9 Since it has been observed that flavonoids, stilbenes, and tropolones manifest tyrosinase inhibitory activities, we have honed our research goals both to evaluate other polyphenolic skeletons for their tyrosinase inhibition properties and also to elucidate their kinetic modes. Broussonetia kazinoki belonging to the family Moraceae in a deciduous tree distributed throughout Korea, China, and Japan. 1,3-Diphenylpropanes, alkaloids, and flavonoids have all been isolated from it.11–17 Broussonetia species have been employed in folk medicine as a diaretic, a tonic, and a suppressant for edema.18,19 Some studies have reported that this species displays cytotoxic, antioxidant and tyrosinase inhibitory activities, but these studies have been relatively resitriced.20–22 For example, although Jang et al. reported the melanogenesis inhibitory activity of paper mulberry, the scope was limited, focusing only on a single extract,

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Y. S. Baek et al. / Bioorg. Med. Chem. 17 (2009) 35–41

kazinol F.22 In addition the authors did not delineate a full kinetic analysis with respect to each oxidation step. In our present study, we have found that bioactivity-guided fractionation of the ethylacetate extracts of Broussonetia kazinoki elicited to new 1,3-diphenolylpropanes, kazinol S and kazinol T, and a known 1,3-diphenylpropanes, kazinol C, kazinol D, kazinol F, and broussonin C. All isolated compounds were additionally evaluated for their inhibitory activities and kinetic modes toward both monophenolase and diphenolase activity of tyrosinase. Interestingly, the most potent compounds examined, 3–5 showed a time-dependent mode.

6.67 ppm. The above mentioned epoxyprenyl group was placed at C-500 on the B-ring due to HMBC correlation of C-500 with H-700 . The 1,1-dimethylallyl group was deduced from the connectivity between H-100 (dH 6.18) and vinyl proton H-110 (dH 5.24–5.32) in COSY spectrum. Further evidence for this group was gleaned from the correlation between C-80 , 90 and H-100 in the HMBC experiment. The position of the 1,1-dimethylally group on the A-ring was determined by the HMBC correlation between H-100 and C50 (dC 124.6). The two isolated protons of H-30 and H-60 in the Aring were observed at 6.28 and 6.93 ppm. Thus, compound 5 was identified as 50 -(2-methylbut-3-en-2-yl)-600 -(3-methylbut-2-enyl)500 -(2,3-epoxy-3-methylbytyl)-20 ,40 ,300 ,400 -tetrahydroxy diphenylpropane called kazinol S (Fig. 2). Analysis of the 13C NMR spectrum disclosed 30 nonequivalent carbon atoms. The high-resolution mass spectrum of this compound revealed a molecular ion m/z 480.2873 consistent with a molecular formula of C30H40O5. This shows that the molecule processes eleven degrees of unsaturation. The 1,1-dimethylallyl group in A-ring and the isoprenyl group in B-ring were deduced by interpretation of NMR spectra which has very similar with the pattern of compound 1. The presence of a dihydrofurano moiety was deduced from the COSY connectivity between methylene protons H-700 (dH 2.98–3.04) and methine proton H-800 (dH 4.61), in conjunction with a HMBC correlation between C-900 (dC 72.9), C-1000 (dC 18.3), C-1100 (dC 30.1) and H-800 . The chemical shifts of C-800 (dC 90.4) and C-900 (dC 72.9) suggested that they were substituted with oxygen to give a 2-(1-hydroxy-1-methylethyl)-dihydrofurano group. The position of dihydrofurano function on B-ring was determined by the HMBC correlation between C-400 (dC 144.5), C-600 (dC 128.1) and H-700 . Thus, compound 6 was identified as 50 -(2-methylbut-3-en-2-yl)-600 -(3-methylbut-2-enyl)-4 00 ,5 00 -[2-(1-hydroxy-1diphenylpropane methylethyl)]-dihydrofuranyl)-20 ,40 ,300 -trihydroxy called kazinol T (Fig. 2).

2. Results and discussion 2.1. Isolation and identification of 1,3-diphneylpropane derivatives Repeated silica gel chromatography of the methanolic extract of the roots of Broussonetia kazinoki yielded six 1,3-diphenylpropanes (Fig. 1). The spectroscopic data of compounds (1–4) agree with those previously published for kazinol C, kazinol D, kazinol F, and broussonin C.11,23 Compound 5 was obtained a yellow oil having the molecular formula C30H40O5 and eleven degrees of unsaturation established by HREIMS (m/z 480.2872 [M+]). The IR spectrum of 5 showed absorptions at 3480, 3050, and 1615 cm1, suggesting the presence of hydroxyl, alkene and aromatic ring moieties. 1H and 13C NMR data in conjunction with DEPT experiments delineated the presence of 30 carbon atoms, consisting of the following functional groups: five methylenes (sp3), one methylene (sp2), five methines (sp2), one methine (sp3), six methyls (sp3), twelve quaternary carbons. The 13C NMR data enabled carbons corresponding to the sixteen C–C double bonds to be identified, and thus accounted for eight of eleven degrees of unsaturation. The extra three degrees of unsaturation were ascribed to two aromatic rings and epoxy ring in diphenylpropane. The presence of isoprenyl group was easily deduced from successive connectivities between H-1200 (dH 3.16) to H-1500 (dH 1.70, CH3, s) and H-1600 (dH 1.65, CH3, s) in 1H-1H COSY spectrum. HMBC correlation of H-1200 with C-600 and C-100 proved the location the isoprenyl moiety. The presence of an epoxyprenyl group was deduced from the connectivity between methylene protons H-700 (dH 2.70 and 2.94) and methane proton H-800 (dH 3.81) in the COSY spectrum and correlation of H-700 with three carbons, C-900 (dC 77.1), 1000 (dC 22.3) and 1100 (dC 24.9) in the HMBC spectrum. The isolated proton of H-200 in B-ring was observed at

9"

16"

2.2. Effects of isolated 1,3-diphenylpropanes on the activity of mushroom tyrosinase In a preliminary screening, using mushroom tyrosinase as a representative enzyme, we observed that methanolic extracts of B. kazinoki roots showed significant inhibition of both L-tyrosine and L-DOPA oxidation. In fractionation, guided by tyrosinase inhibitory activity, EtOAc and MeOH fractions from methanol extracts showed strong inhibitory activity against mushroom tyrosinase. More detailed bioassay of the isolated compounds were subse-

11"

7" 3'

HO 10' 11'

7'

OH

OH

12"

5'

1'

1

9'

3

HO

OH

O

HO

OH

OH

B

A 1"

2"

OH

OH

1

OH

2

3

OH

O HO

OH

OH

HO

OH

OH

HO

O

OH

OH

4

5 Figure 1. Chemical structures of isolated compounds 1–6 from the Broussonetia kazinoki.

OH

6

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Y. S. Baek et al. / Bioorg. Med. Chem. 17 (2009) 35–41 9"

16"

11"

OH

O

14" 7" 3'

HO 5'

OH

HO

O

OH

3"

1'

7'

11'

OH 12" 3

OH

1

OH

2" 9'

5

6

Figure 2. Selected HMBC correlations for new compounds 5 and 6.

Table 1 Inhibitory effects of isolated compounds 1–6 on mushroom tyrosinase activities Compound

1 2 3 4 5 6

L-Tyrosine

L-DOPA

IC50a (lM)

Type of inhibition (Ki, lM)b

IC50 (lM)

Type of inhibition (Ki, lM)

15.5 164.7 0.96 0.43 17.9 103.1

Competitive NTc Competitive Competitive Competitive NT

22.8 >200 1.7 0.57 26.9 >200

Competitive NT Competitive Competitive Competitive NT

(10.8) (0.41) (0.23) (9.12)

(11.2) (0.77) (0.29) (15.6)

a All compounds were examined in a set of experiments repeated three times; IC50 values of compounds represent the concentration that caused 50% enzyme activity loss. b Values of inhibition constant. c Not tested.

quently conducted. As shown in Table 1, all 1,3-diphenylpropanes examined, apart from compounds 2 and 6 exhibited dose-dependent inhibitory effect on the monophenolase activity of mushroom tyrosinase (IC50 0.43–17.9 lM). The same compounds also appreciably inhibited diphenolase with IC50 values of 22.8, 1.7, 0.57, and 26.9 lM, respectively. As the concentrations of the inhibitors were augmented, the residual enzyme activity drastically diminished (Fig. 3A). All inhibitors manifested a similar relationship between enzyme activity and enzyme concentration. The inhibition of mushroom tyrosinase by compound 1 is illustrated in Figure 3B, representatively. Plots of residual enzyme activity versus enzyme concentration at different concentrations of compound 1 gave a family of straight lines with a y-axis intercept of 0, consistent with 1 being reversible inhibitor. Increasing concentration of compound 1 resulted in a reduction of the slopes of the lines. Taken as an ensemble, the following general features of the SAR can be deduced from compounds 1–6. The presence of a free

Comp. 1 Comp. 3 Comp. 4 Comp. 5

80

2.3. Time-dependent inhibitory effects of 1,3diephenylpropanes on mushroom tyrosinase The time dependence of the isolated compounds on the tyrosinase-catalyzed oxidation of L-DOPA were subsequently investigated. These values were compared to a typical diphenolase inhibitor. Compounds (1, 3–5) showed a typical progress curve of time-dependent inhibition. As described below (Fig. 4A). For example, the most potent tyrosinase inhibitor, compound 4, showed a typical progress curve for slow-binding inhibition at low concentrations (Fig. 4B and C). Slow-binding inhibition mechanisms are generally investigated by preincubation of the enzyme with inhibitor over various time points prior to measurement of initial velocities for substrate oxidation as a function of preincubation time. This allows the amount of residual active enzyme to be measured. The kobs values for the inhibition of tyrosinase at different concentrations of compound 4 were determined by fitting data to the slow-binding equation (Eq. 3). kobs was then plotted as a function of inhibitor concentrations. The inhibition was shown to be time dependent by the fact that kobs exhibited a liner dependence on

B

60 40

0.25

0 μM 5 μM 10 μM 15 μM

0.20 ΔA475nm

Tyrosinase activity (%)

A 100

hydroxyl group, a component of a 1,2-dihydroxyphenol motif at C-400 is important for activity. Compounds lacking this motif showed IC50s around 10-fold less than those thus equipped, for example, 2 (IC50 = 164.7 lM) vs 5 (IC50 = 17.9 lM). However, an effect of great magnitude was observed by removing alkyl substitution at C-50 , for example, 1 (IC50 = 15.5 lM) versus 4 (IC50 = 0.43 lM). This effect may be accounted for by unfavorable steric interactions between the C-50 function and the active site, either by simply augmenting the size of the A-ring, or by impending H-bonding to OH at C-40 . Finally, a comparison of the two most potent inhibitors, 3 and 4, unveils that additional substitution in the A-ring has little effect on the inhibitor potency (IC50 0.96 lM vs 0.43 lM, respectively).

0.15 0.10 0.05

20 0

0.00 0.01

0.1

1 [I], (μM)

10

100 1000

0

100

200

300

400

500

Enzyme units

Figure 3. (A) Effect of compounds (1, 3, 4, and 5) on the activity of tyrosinase for the oxidation of L-DOPA. (B) The catalytic activity of tyrosinase as function of enzyme concentrations at different concentrations of compound 1 (d, 0 lM; s, 5 lM; ., 10 lM; 4, 15 lM).

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Y. S. Baek et al. / Bioorg. Med. Chem. 17 (2009) 35–41

A

0.5

B

0

0.5

0 1

0.4

0.4 4

0.2

2

0.1

3

ΔA475nm

ΔA475nm

1

0.3

2

0.2 3

0.1 0.0

0.0 0

C

0.3

1

2 3 Time (min)

4

0

5

D

e0

1

2 3 Time (min)

4

5

0.10

0

0.08

-1 (m )

0.06 0.04

obs.

v /v 0

2

kobs. (m -1)

1

k

3

0.02

0.06 0.04 0.02 0.00 0.0

0.2

0.4

0.6

4 (μM)

0.00 0

2

4

6

8

10

0

Preincubation time (min)

5

10

15

20

[I], (μM)

Figure 4. (A) Time course of oxidation of L-DOPA catalyzed by mushroom tyrosinase in the presence of compounds (1, 3, 4, and 5). Concentrations of compounds 1 (s), compounds 3 (.), compounds 4 (4), and compounds 5 (j) were 20 lM, 3 lM, 2 lM, and 20 lM. (B) Time-dependent inhibition of tyrosinase in the presence of compound 4, representatively. Concentrations of compound 4 from top to bottom were 0, 1, 1.5, and 2 lM. (C) Preincubation time dependence of the fractional velocity of an enzymecatalyzed reaction in the presence of varying concentrations of a compound 4 on a semilog scale (d, 0.3 lM; s, 0.3 lM; ., 0.4 lM; 4, 0.6 lM). (D) Plot of kobs as a function of inhibitors (3–5) concentration for a slow-binding inhibitor fitted by Eq. 3 (d, 3; s, 4; ., 5).

the inhibitor concentration as depicted in Figure 4D. Thus, these results prove that 1,3-diphenylpropanes inhibit tyrosinase by the simple reversible slow-binding model according to analysis by Eqs. 3 and 4 yielded the data in Table 2. Recently, many researchers reported that 4-substituted resorcinols such as 4-ethylresorcinol, 4-hyxylresorcinol and 4-dodecylresorcinol can be classified as slow-binding competitive inhibitors of mushroom tyrosinase.24 In this study, we isolated six 1,3-diphenylpropanes, exhibiting a resorcinol function in the A-ring. Consistent with the structural similarities to the inhibitors employed in the above results, our inhibitors also show time-dependent inhibition behavior. 2.4. Determination of the inhibition type of 1,3diphenylpropanes on mushroom tyrosinase The kinetic behaviors of the oxidation of L-tyrosine and L-DOPA, catalyzed by mushroom tyrosinase at different concentrations of compounds (1, 3–5) were studied. In this experiment, the initial velocity of the enzyme was monitored via dopachrome formation at 475 nm. As shown in Figure 5, the Lineweaver–Burk plots of

Table 2 Kinetic parameters for time-dependent inhibitor of tyrosinase by compounds (3–5) 1

Compound

k3 (lM

3 4 5

0.0301 0.0993 0.0228

min

1

)

1

k4 (min 0.0054 0.0048 0.0197

)

Ki

app

(lM)

0.1808 0.0485 0.8696

1/V versus 1/[S] result in a family of straight lines with the same y-axis intercept, as illustrated, respectively, for the four tyrosinase inhibitors. In the figure, the abscissa 1/[L-DOPA] is the reciprocal of the concentrations of L-tyrosine, whereas the ordinate 1/V is the reciprocal of the change of the velocity, which reflects the reciprocal of tyrosinase activity. The arrangement of the family of lines in each graph indicated that they are all are competitive inhibitors [(1, Ki = 11.2 lM), (3, 0.77 lM), (4, 0.29 lM), and (5, 15.6 lM)], with their inhibitory activity toward tyrosinase decreasing with increasing concentration of the substrate. Most competitive inhibitors of tyrosinase have molecular structures which closely resemble that of the product of each respective step. Nerya and his coworkers reported that the OH group substation at para position of the chalcone benzene ring is the major factor affecting inhibitory potency, because it results in a molecular skeleton closely similar to that of tyrosine.25 With respect to diphenolases activity, L-DOPA, an o-diphenol, is the substrate. For successful diphenolase inhibition, a 3,4-dihydroxy group in the B-ring of required in order to make the structure of the inhibitor molecule resemble L-DOPA. This leads to the competitive displacement of L-DOPA from the active site of the cofactor in a lock-andkey model. This is also seen in molecules such as quercetin.26 3. Conclusion Six 1,3-diphenylpropane derivatives were successfully isolated and purified from B. kazinoki including new 1,3-diphenylpropanes 5 and 6, which we named ‘kazinol S’ and ‘kazinol T’. We assayed the inhibitory properties of these species against mono- and

Y. S. Baek et al. / Bioorg. Med. Chem. 17 (2009) 35–41

15

B 0 μM 10.0 μM 20.0 μM 30.0 μM

10

14 12 1/v (μmole/min)

1/v (μmole/min)

A 20

39

5

10

0 μM 1.0 μM 1.5 μM 2.0 μM

8 6 4 2

1/[L-DOPA], (μM)

C

14

1/v (μmole/min)

12 10

0 μM 0.3 μM 0.5 μM 0.75 μM

8 6 4

-0.002 0.000 0.002 0.004 0.006 0.008 1/[L-DOPA], (μM)

D 1/v (μmole/min)

-0.002 0.000 0.002 0.004 0.006 0.008

20

15

0 μM 20.0 μM 30.0 μM 40.0 μM

10

5

2 -0.002 0.000 0.002 0.004 0.006 0.008 1/[L-DOPA] , (μM)

-0.002 0.000 0.002 0.004 0.006 0.008 1/[L-DOPA], (μM)

Figure 5. Lineweaver–Burk plots for the inhibition of the diphenolase activity of tyrosinase by compounds (1, 3, 4, and 5). (A) Concentrations of compound 1 for curves from top to bottom were 0, 10, 20, and 30 lM; (B) compound 3 (0, 1, 1.5, and 2 lM); (C) compound 4 (0, 0.3, 0.5, and 0.75 lM), and (D) compound 5 (0, 20, 30, and 40 lM), respectively.

diphenolases activity of tyrosinase. Four of these compounds emerged to be potent inhibitors of both these processes. We progressed to clearly demonstrate that all these species were competitive inhibitors as well as time dependent. It is our strong belief that these data will be of use in the continuing fight against tyrosinase-born illness and food depreciation. 4. Materials and methods 4.1. Materials Chromatographic separations were carried out by Thin-layer Chromatography (TLC) (E. Merck Co., Darmastdt, Germany), using commercially available glass plate pre-coated with silica gel and visualized under UV at 254 and 366 nm sprayed with p-anisaldehyde staining reagent. Column chromatography was carried out using 230–400 mesh silica gel (kieselgel 60, Merck, Germany). Silica gel (230-400 mesh, Merck), RP-18 (ODS-A, 12 nm, S-150 mM, YMC), and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography. Melting points were measured on a Thomas Scientific capillary melting point apparatus (Electrothermal 9300, UK) and are uncorrected. IR spectra were recorded on a Bruker IFS66 (Bruker, karlsruhe, Germany) infrared Fourier transform spectrophotometer (KBr) and UV spectra were measured on a Beckman DU650 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). 1H and 13C NMR along with 2D NMR data were obtained on a Bruker AM 500 (1H NMR at 500 MHz, 13C NMR at 125 MHz) spectrometer (Bruker, karlsruhe, Germany) in CDCl3, and acetone-d6 with TMS as internal standard. EIMS was obtained on a JEOL JMS-700 mass spectrometer (JEOL, Tokyo, Japan). Qualitative

analyses were measured on a Perkin-Elmer HPLC S200 (Perkin-Elmer, CA, USA). All the reagent grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 4.2. Isolation of tyrosinase inhibitors from B. kazinoki The roots of barks (3 kg) of Broussonetia kazinoki were air-dried, chopped and extracted three times with methanol (2 18 L) for 10 days at room temperature. The combined methanol extract was concentrated in vacuo to yield a dark brown gum (89 g). Prior to vacuum liquid chromatography (VLC), the methanol extract was successively partitioned with hexane, EtOAc, and MeOH (each 10 L), yielding a hexane extract (12 g), a EtOAc extract (49 g), and a MeOH extract (21 g). The EtOAc phase was chromatographed on silica gel (6 60 cm, 230–400 mesh, 800 g) using hexane/EtOAc [30:1 (1.5 L), 20:1 (1.5 L), 10:1 (1.5 L), 5:1 (1.5 L), 3:1 (1.5 L), 1:1 (3 L)] mixtures to give fraction F1–F6. Fraction F2 (2.3 g) and silica gel (230–400 mesh, 4 g) was dissolved in a minimum volume of CHCl3 and concentrated under vacuum. This precoated resin was loaded onto the top of grass column containing silica gel (230– 400 mesh, 80 g) and chromatographed with hexane/EtOAc (40:1 ? 1:1) to afford 5 subfractions (F2.1–F2.5); subfraction F2.3 was rechromatographed using the same solvent gradient, to yield compound 6 (15 mg). Fraction F3 (12.1 g) was applied to a silica gel column (3 60 cm, 230–400 mesh, 120 g) and chromatographed with hexane/EtOAc (20:1 ? 1:1) to afford 7 subfractions (F3.1–F3.7); subfractions (F3.3–F3.4) were purified using sephadex LH-20 column chromatography, eluting with 100% MeOH to afford compound 1 (52 mg) and compound 2 (34 mg). Fraction F4–F5 were subjected to silica gel column (4 50 cm, 230–400 mesh,

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Y. S. Baek et al. / Bioorg. Med. Chem. 17 (2009) 35–41

250 g) chromatographed with CHCl3/MeOH (99:1 ? 2:1) and then purified by reversed-phase column chromatography. Thus, this sample was loaded onto a grass column packed with RP-18 (ODS-A, 12 nm, S-150 lM, 40 g). The column was then eluted using MeOH: H2O (4:1) to afford compound 3 (22 mg), compound 4 (20 mg), and compound 5 (12 mg). All of isolated compounds were identified on the basis of the following spectroscopic data. Compound 1. Obtained as a oily substance; 1H NMR (500 MHz, CDCl3) d 1.37 (6 H, s, CH3-80 , CH3-90 ), 1.65 (3H, s, CH3-1000 ), 1.68 (3H, s, CH3-1100 ), 1.69 (3H, s, CH3-1500 ), 1.77 (3H, s, H-160 0 ), 1.80 (2H, m, H-2), 2.52 (2H, m, H-3), 2.57 (2H, m, H-1), 3.21 (2H, d, J = 5.8 Hz, H-1200 ), 3.32 (2H, d, J = 6.5 Hz, H-700 ), 4.95 (1H, m, H1300 ), 5.11 (1H, m, H-800 ), 5.24 (1H, d, J = 17.6 Hz, H-110 ), 5.26 (1H, d, J = 11.5Hz, H-110 ), 6.14 (1H, dd, J = 17.6, 10.5 Hz, H-100 ), 6.29 (1H, s, H-30 ), 6.58 (1H, s, H-200 ), 6.93 (1H, s, H-60 ). 13C NMR (125 MHz, CDCl3) d 33.3 (C-1), 30.3 (C-2), 31.9 (C-3), 120.9 (C-10 ), 153.5 (C-20 ), 105.3 (C-30 ), 153.6 (C-40 ), 124.7 (C-50 ), 128.0 (C-60 ), 40.2 (C-70 ), 27.6 (C-80 , 90 ), 148.8 (C-100 ), 113.6 (C-110 ), 133.4 (C100 ), 114.4 (C-200 ), 142.1 (C-30 0 ), 140.8 (C-400 ), 130.7 (C-500 ), 127.2 (C-600 ), 28.1 (C-700 ), 123.0 (C-800 ), 133.8 (C-900 ), 18.4 (C-1000 ), 26.1 (C-1100 ), 26.5 (C-1200 ), 124.5 (C-1300 ), 131.2 (C-1400 ), 18.3 (C-1500 ), 26.0 (C-1600 ). Compound 2. Obtained as a oily substance; 1H NMR (500 MHz, CDCl3) d 1.31 (6H, s, CH3-1000 , CH3-1100 ), 1.39 (6H, s, CH3-80 , CH390 ), 1.66 (3H, s, CH3-1600 ), 1.71 (3H, s, CH3-1500 ), 1.81 (2H, m, H800 ), 1.85 (2H, m, H-2), 2.55 (2H, m, H-3), 2.59 (2H, m, H-1), 2.65 (2H, m, H-70 0 ), 3.18 (2H, d, J = 5.8 Hz, H-1200 ), 4.94 (1H, m, H-1300 ), 5.24 (1H, d, J = 17.6 Hz, H-110 ), 5.26 (1H, d, J = 11.5 Hz, H-110 ), 6.15 (1H, dd, J = 17.6, 10.5 Hz, H-100 ), 6.29 (1H, s, H-30 ), 6.65 (1H, s, H-200 ), 6.94 (1H, s, H-60 ). 13C NMR (125 MHz, CDCl3) d 33.0 (C1), 29.9 (C-2), 32.0 (C-3), 120.5 (C-10 ), 153.8 (C-20 ), 105.1 (C-30 ), 153.6 (C-40 ), 124.6 (C-50 ), 127.8 (C-60 ), 40.1 (C-70 ), 27.6 (C-80 ,90 ), 148.8 (C-100 ), 113.2 (C-110 ), 132.1 (C-100 ), 113.5 (C-200 ), 143.4 (C300 ), 139.5 (C-400 ), 120.1 (C-500 ), 129.3 (C-600 ), 20.6 (C-700 ), 33.6 (C800 ), 74.5 (C-900 ), 27.1 (C-1000 ), 27.1 (C-1100 ), 26.7 (C-120 0 ), 123.8 (C1300 ), 131.5 (C-1400 ), 18.3 (C-1500 ), 26.0 (C-1600 ). Compound 3. Crystallized from benzene to give colorless needles, mp 108–109 °C; 1H NMR (500 MHz, acetone-d6) d 1.51 (3H, s, CH3-1000 ), 1.52 (3H, s, CH3-1100 ), 1.57 (3H, s, CH3-1600 ), 1.59 (3H, s, CH3-1500 ), 1.64 (2H, m, H-2), 2.34 (2H, m, H-3), 2.45 (2H, m, H1), 3.08 (2H, d, J = 5.8 Hz, H-1200 ), 3.21 (2H, d, J = 6.5 Hz, H-70 0 ), 4.82 (1H, m, H-1300 ), 4.96 (1H, m, H-800 ), 6.12 (1H, dd, J = 8.1, 2.3 Hz, H-50 ), 6.24 (1H, d, J = 2.3 Hz, H-30 ), 6.42 (1H, s, H-20 0 ), 6.74 (1H, d, J = 8.1 Hz, H-60 ). 13C NMR (125 MHz, acetone-d6) d 34.0 (C-1), 31.0 (C-2), 33.4 (C-3), 120.9 (C-10 ), 157.0 (C-20 ), 103.8 (C30 ), 157.7 (C-40 ), 107.6 (C-50 ), 131.5 (C-60 ), 40.2 (C-70 ), 133.0 (C100 ), 114.9 (C-200 ), 143.3 (C-300 ), 142.5 (C-400 ), 130.8 (C-500 ), 128.1 (C-600 ), 26.6 (C-70 0 ), 125.3 (C-80 0 ), 131.1 (C-90 0 ), 18.5 (C-1000 ), 26.2 (C-1100 ), 28.5 (C-1200 ), 126.1 (C-1300 ), 131.1 (C-1400 ), 18.4 (C-1500 ), 26.2 (C-1600 ). Compound 4. Obtained as a oily substance; 1H NMR (500 MHz, acetone-d6) d 1.56 (6H, s, CH3-1000 , CH3-1100 ), 1.70 (2H, m, H-2), 2.38 (2H, m, H-3), 2.41 (2H, m, H-1), 3.16 (2H, d, J = 7.2 Hz, H700 ), 5.21 (1H, m, H-80 0 ), 6.12 (1H, dd, J = 8.1, 2.2 Hz, H-30 ), 6.24 (1H, d, J = 2.0 Hz, H-50 ), 6.59 (1H, d, J = 8.0 Hz, H-200 ), 6.70 (1H, dd, J = 8.0, 2.0 Hz, H-600 ), 6.74 (1H, d, J = 8.1 Hz, H-60 ), 6.78 (1H, d, J = 1.9 Hz, H-30 0 ). 13C NMR (125 MHz, acetone-d6) d 36.0 (C-1), 33.6 (C-2), 30.4 (C-3), 120.9 (C-10 ), 157.0 (C-20 ), 107.6 (C-30 ), 157.7 (C-40 ), 103.8 (C-50 ), 132.3 (C-60 ), 128.7 (C-100 ), 115.9 (C-200 ), 130.7 (C-300 ), 154.0 (C-400 ), 134.7 (C-500 ), 127.6 (C-600 ), 29.5 (C-700 ), 124.5 (C-800 ), 132.4 (C-900 ), 18.2 (C-100 0 ), 26.3 (C-1100 ). Compound 5. Obtained as a oily substance; 1H NMR (500 MHz, CDCl3) d 1.30 (3H, s, CH3-1100 ), 1.34 (3H, s, CH3-1000 ), 1.38 (6H, s, CH3-80 , CH3-90 ), 1.65 (3H, s, CH3-1600 ), 1.70 (3H, s, CH3-1500 ), 1.83 (2H, m, H-2), 2.54 (2H, m, H-3), 2.58 (2H, m, H-1), 2.70 (1H, dd, J = 17.0, 5.4 Hz, H-700 a), 2.94 (1H, dd, J = 17.0, 5.0 Hz, H-700 b), 3.16

(2H, d, J = 5.9 Hz, H-1200 ), 3.81 (1H, m, H-800 ), 4.90 (1H, m, H-1300 ), 5.25 (1H, d, J = 10.6 Hz, H-110 ), 5.32 (1H, d, J = 17.6 Hz, H-110 ), 6.18 (1H, dd, J = 17.6, 10.5 Hz, H-100 ), 6.28 (1H, s, H-30 ), 6.67 (1H, s, H-20 0 ), 6.93 (1H, s, H-60 ). 13C NMR (125 MHz, CDCl3) d 29.8 (C1), 31.9 (C-2), 33.0 (C-3), 120.5 (C-10 ), 153.8 (C-20 ), 105.1 (C-30 ), 153.6 (C-40 ), 124.6 (C-50 ), 127.9 (C-60 ), 40.1 (C-70 ), 27.5 (C-80 , 90 ), 148.8 (C-100 ), 113.5 (C-110 ), 133.4 (C-100 ), 113.9 (C-200 ), 138.2 (C300 ), 143.3 (C-400 ), 118.3 (C-500 ), 129.9 (C-600 ), 29.9 (C-700 ), 70.6 (C800 ), 77.1 (C-900 ), 22.3 (C-1000 ), 24.9 (C-1100 ), 27.7 (C-1200 ), 123.4 (C1300 ), 131.9 (C-1400 ), 18.4 (C-1500 ), 24.9 (C-1600 ). Compound 6. Obtained as a oily substance; 1H NMR (500 MHz, CDCl3) d 1.17 (3H, s, H-1600 ), 1.30 (3H, s, CH3-1500 ), 1.38 (6H, s, CH3-80 , CH3-90 ), 1.65 (3H, s, CH3-1100 ), 1.68 (3H, s, CH3-1000 ), 1.80 (2H, m, H-2), 2.52 (2H, m, H-3), 2.57 (2H, m, H-1), 3.04 (2H, m, H-700 ), 3.12 (2H, d, J = 6.2 Hz, H-1200 ), 4.61 (1H, m, H800 ), 4.98 (1H, m, H-1300 ), 5.23 (1H, d, J = 17.6 Hz, H-110 ), 5.29 (1H, d, J = 10.5 Hz, H-110 ) 6.14 (1H, dd, J = 17.6, 10.5 Hz, H-100 ), 6.29 (1H, s, H-30 ), 6.58 (1H, s, H-200 ), 6.93 (1H, s, H-60 ). 13C NMR (125 MHz, CDCl3) d 29.1 (C-1), 32.1 (C-2), 32.4 (C-3), 120.6 (C-10 ), 153.7 (C-20 ), 105.2 (C-30 ), 153.7 (C-40 ), 124.5 (C50 ), 127.9 (C-60 ), 40.1 (C-70 ), 27.6 (C-80 , 90 ), 148.8 (C-100 ), 113.3 (C-110 ), 127.5 (C-100 ), 116.8 (C-200 ), 137.9 (C-300 ), 144.5 (C-400 ), 134.0 (C-500 ), 128.1 (C-600 ), 29.1 (C-700 ), 90.4 (C-800 ), 72.9 (C-900 ), 18.3 (C-100 0 ), 30.1 (C-1100 ), 31.3 (C-1200 ), 123.2 (C-1300 ), 131.6 (C-1400 ), 26.3 (C-1500 ), 23.6 (C-160 0 ). 4.3. Assay of tyrosinase activity Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was used as described previously with some modifications, using either, L-DOPA (diphenolase) or L-tyrosine (monophenolase) as substrate.10 In spectrophotometric experiments, enzyme activity was monitored by observing dopachrome formation at 475 nm with a UV–Vis spectrophotometer (Spectro UV–vis Double beam; UVD-3500, Labomed, Inc.) at 30 °C. All samples were first dissolved in EtOH at 10 mM. First, 200 lL of a 2.7 mM L-tyrosine (Km = 180 lM) or 5.4 mM L-DOPA (Km = 360 lM) aqueous solution was mixed with 2687 lL of 0.25 M phosphate buffer (pH 6.8). Then, 100 lL of the sample solution and 13 lL of the same phosphate buffer solution containing mushroom tyrosinase (144 units) were added in this order to the mixture. Each assay was conducted as three separate replicates. The inhibitor concentration leading to 50% activity loss (IC50) was obtained by fitting experimental data to the logistic curve by Eq. 127:

Activity ð%Þ ¼ 100½1=ð1 þ ð½I=IC50 ÞÞ

ð1Þ

4.4. Progress curves determination and time-dependent assay All reactions were carried out using L-tyrosine as a substrate in 0.25 M phosphate buffer (pH 6.8) at 30 °C. Enzyme activities were measured continuously for 15 min using a UV spectrophotometer. To determine the kinetic parameters associated with time-dependent inhibition of tyrosinase, progress curves with 20 data points (30 s intervals) were obtained at several inhibitor concentrations using fixed substrate concentrations. A plot of the natural log of the residual enzyme activity versus preincubation time gave a straight line with a slope of kobs. The values of k3, k4, and Kiapp were calculated from the plot of the kobs versus concentration of inhibitors according to the method of Morrison and Walsh.28–30 The data were analyzed using the a nonlinear regression program [Sigma Plot (SPCC Inc., Chicago, IL)] to give the individual parameters for each curve; vi (initial velocity), vs (steady-state velocity), kobs (apparent first-order rate constant for the transition from vi to vs), A (absorbance at 475 nm), and Kiapp (apparent Ki) according to the following equations27–30:

Y. S. Baek et al. / Bioorg. Med. Chem. 17 (2009) 35–41

A ¼ v s t þ ðv i  v s Þ½1  expðkobs tÞ=kobs

ð2Þ

v =v 0 ¼ expðkobs tÞ

ð3Þ

Þ kobs ¼ k4 ð1 þ ½I=K app i

ð4Þ

Acknowledgments This work was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare (A080813) and the MOST/KOSEF to the Environmental Biotechnology National Core Research Center (R15-2003-012-02001-0), Republic of Korea. Y.S. Baek was supported by a scholarship from the BK21 program, the Ministry of Education and Human Resources Development, Republic of Korea. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmc.2008.11.022. References and notes 1. 2. 3. 4.

Fu, B.; Li, H.; Wang, X.; Lee, F. S. C.; Cui, S. J. Agric. Food Chem. 2005, 53, 7408. Meada, K.; Fukuda, M. J. Soc. Cosmet. Chem. 1991, 42, 361. Mcevily, J. A.; Iyengar, R.; Otwell, Q. S. Crit. Rev. Food Sci. Nutr. 1992, 32, 253. Xu, Y.; Stokes, A. H.; Freeman, W. M.; Kumer, S. C.; Vogt, B. A.; Vrana, K. E. Mol. Brain Res. 1997, 45, 159. 5. Halaouli, S.; Asther, M.; Sigoillot, J. C.; Hamdi, M.; Lomascolo, A. J. Appl. Microbiol. 2006, 100, 219. 6. Mayer, A. M. Phytochemistry 2006, 67, 2318.

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7. Fenoll, L. G.; Penalver, M. J.; Rodriguez-Lopez, J. N.; Varon, R.; Garcia-Canovas, F.; Tudela, J. Int. J. Biochem. Cell Biol. 2004, 36, 235. 8. Kubo, I.; Nihei, K. I.; Shimizu, K. Bioorg. Med. Chem. Lett. 2004, 12, 5343. 9. Matsuura, R.; Ukeda, H.; Sawamura, M. J. Agric. Food Chem. 2006, 54, 2309. 10. Masamoto, Y.; Iida, S.; Kubo, M. Planta Med. 1980, 40, 361. 11. Ikuta, J.; Hano, Y.; Nomura, T.; Kawakami, Y.; Sato, T. Chem. Pharm. Bull. 1986, 34, 1968. 12. Kato, S.; Fukai, T.; Kawakami, Y.; Sato, T. Chem. Pharm. Bull. 1986, 34, 2448. 13. Shibano, M.; Nakamura, S.; Kubori, M.; Minoura, K. Chem. Pharm. Bull. 1998, 46, 1416. 14. Shibano, M.; Kitagawa, S.; Nakamura, S.; Akazawa, N.; Kusano, G. Chem. Pharm. Bull. 1997, 45, 700. 15. Tsukamoto, D.; Shibano, M.; Okamoto, R.; Kusano, G. Chem. Pharm. Bull. 2001, 49, 492. 16. Barron, D.; Ibrhim, R. Phytochemistry 1996, 43, 921. 17. Zhang, P. C.; Wnag, S.; Wu, Y.; Chen, R. Y.; Yu, D. Q. J. Nat. Prod. 2001, 64, 1206. 18. Ryu, J. H.; Ahn, H.; Lee, H. J. Fitoterapia 2003, 74, 350. 19. ‘Zhong Yao Da Ci Dian,’ Vol. 2, ed. by Jian Su New Medical, Shanghai, 1977, p 1502. 20. Ko, H. H.; Yen, M. H.; Wu, R. R.; Won, S. J.; Lin, C. N. J. Nat. Prod. 1999, 62, 164. 21. Lee, H. J.; Park, J. H.; Jang, D. I.; Ryu, J. H. Yakhak Hoechi 1997, 41, 439. 22. Jang, D. I.; Lee, B. G.; Jeon, C. O.; Jo, N. S.; Park, J. H.; Cho, S. Y.; Lee, H.; Koh, J. S. Cosmet. Toilet. 1997, 112, 59. 23. Takasugi, M.; Kumagai, Y.; Nagao, S.; Masamune, T.; Shirata, A.; Takahashi, K. Chem. Lett. 1980, 1459. 24. Mercedes, J.; Francisco, G. C. J. Agric. Food Chem. 1997, 45, 2061. 25. Ohad, N.; Ramadan, M.; Soliman, K.; Snait, T.; Jacob, V. Phytochemistry 2004, 65, 1389. 26. Kubo, I.; Ikuyo, K. H.; Chaudhuri, S. K.; Kubo, Y.; Sanchez, Y.; Ogurab, T. Bioorg. Med. Chem. 2000, 8, 1749. 27. Copeland, R. A. Enzyme: A Practical Introduction to Structure, Mechanism, and Data Analysis; Wiley-VCH: New York, 2000. 28. Frieden, C. J. Biol. Chem. 1970, 245, 5578. 29. Morrison, J. F.; Walsh, C. T. Adv. Enzymol. 1988, 61, 201. 30. Sculley, M. J.; Morrison, J. F.; Cleland, W. W. Biochimi. Biophys. Acta Protein Struct. Mol. Enzymol. 1996, 1298, 78.