Natural Product Sciences

17(3) : 198-201 (2011)

Tyrosinase Inhibitory Constituents of Morus bombycis Cortex†

Kyo Bin Kang1, Sang Du Kim1, Tae Bum Kim1, Eun Ju Jeong1, Young Choong Kim1,
Jong Hyuk Sung2, and Sang Hyun Sung1*
College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University,
1

Daehak-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea

2
CHA stem cell institutes, CHA University, 606-16 Yeoksam-Dong, Gangnam-Gu, Seoul 135-080, Korea

Abstract − Tyrosinase is one of the important enzymes in the mammalian melanin synthesis. In the process of
melanin synthesis, tyrosine is oxidized to DOPA (3,4-dihydroxyphenylalanine), and DOPA is further oxidized to
dopaquinone. Tyrosinase is an enzyme catalyzing this oxidation of tyrosine, so chemicals that inhibit the activity
of tyrosinase can be used as skin whitening agents. In this study, we isolated five constituents from the 80%
MeOH extract of Morus bombycis cortex by bioactivity-guided fractionation. We performed mushroom tyrosinase
inhibition assay. As a result, 7,2',4'-trihydroxyflavanone (1), 2',4',2,4,-tetrahydroxychalcone (2), and oxyresveratrol
(3) showed the more potent inhibitory effect compared to kojic acid, a well-known skin whitening agent with anti-
tyrosinase effect. Moracinoside M (4) and moracin M-3'-O-β-D-glucopyranoside (5) also showed the moderate
tyrosinase inhibitory activities.
Keywords − Morus bombycis, skin whitening, tyrosine, tyrosinase inhibitory effect

Introduction Recently, the cortex of Morus species has been shown

to exhibit skin-whitening effect (Lee et al., 2002).
Tyrosinase (monophenol, dihydroxy-L-phenylalanin: Cosmetics with skin-whitening effect which contain the
oxygen oxidoreductase, EC 1.14.18.1) is a copper- extract of Morus alba or Morus bombycis root bark are
dependent protein widely distributed in nature (van released and received favorable responses on market.
Gelder et al, 1997). It is the key enzyme responsible for Even though oxyresveratrol isolated from Morus has been
enzymatic browning of many plant-derived food products suggested to perform the major role in inhibiting
(Mayer, 1987). It also contributes to the formation of tyrosinase followed by generating skin-whitening effect,
melanin pigments in mammals (Slominski et al, 2004). the research on the bio-active constituents of this plants is
Tyrosinase catalyzes two different reactions: the hydroxy- still limited (Shin et al., 1998; Lee et al., 2002). In this
lation of monophenols to o-diphenols (monophenolase study, we isolated a number of constituents from Morus
activity) and the oxidation of odiphenols to o-quinones bombycis cortex by bioactivity-guided fractionation, and
(diphenolase activity), both utilizing molecular oxygen evaluated the inhibitory effects of the isolated compounds
(Siegbahn, 2003). on mushroom tyrosinase.
These two reactions take place in the formation of
mammalian melanin biosynthesis. The biosynthetic steps Experimental
for the formation of melanin include the hydroxylation of
l-tyrosine to 3-4-dihydroxyphenylalanine (l-dopa) and the General Experimental Procedures − All solvents
oxidation of l-dopa to o-dopaquinone (Solano et al., 2006; were purchased from Daejung Chemicals & Metals Co.
Martnez-Esparza et al., 1998). Tyrosinase catalyzes these Ltd., Korea, unless stated otherwise. All solvents for
two hydroxylation reactions, therefore, the inhibitors of HPLC were purchased from Fisher Scientific Korea
tyrosinase are thought to have potential as skin-whitening Ltd., Korea. Kiesgel 60 (40 - 60 µm, 230 - 400 mesh,
agents. Art. 9385, Merck, Germany) was used for the silica gel
column chromatography. TLC was performed using
†
Dedicated to Prof. Young Choong Kim of the Seoul National Univer- Merck precoated silica gel F plates and RP-18 F
sity for her leading works on Pharmacognosy 254 254

*Author for correspondence plates. Ultrasonication was performed using a Branson

Tel: +82-2-880-7859; E-mail: [email protected]

198
Vol. 17, No. 3, 2011 199

5200 sonicator. MPLC was performed using Teledyne 2.3 Hz, H-6), 6.42 (1H, d, J = 1.9 Hz, H-3'), 6.39 (1H, dd,
CombiFLASH RETRIEVE using Teledyne Redisep Rf J = 2.3, 8.3 Hz, H-5'), 6.38 (1H, d, J = 2.3 Hz, H-8), 5.69
C18 reverse phase columns. Preparative HPLC was (1H, dd, J = 13.3, 2.7 Hz, H-2), 3.02 (1H, dd, J = 16.7,
performed using HITACHI L-7100 pump with HITACHI 15.4 Hz, H-3eq.), 2.65 (1H, dd, J = 16.7, 2.8 Hz, H-3ax.).
L-7420 UV detector and Phenomenex Hemini C18 5 µm 13
C-NMR (125 MHz, acetone-d6): δ 191.7 (C-4), 165.6
column (150 × 10 mm). ESI MS data were obtained on (C-9), 165.4 (C-7), 159.8 (C-4'), 156.6 (C-2'), 130.0 (C-
Agilent 1100 series LC/MSD trap LC/MS. NMR spectra 5), 129.4 (C-6'), 118.6 (C-1'), 115.6 (C-10), 111.5 (C-6),
were recorded on JEOL GSX 400 Spectrometer (400 108.3 (C-5'), 104.1 (C-8), 103.9 (C-3'), 76.3 (C-2), 44.2
MHz), JEOL LA 300 Spectrometer (300 MHz), and (C-3).
Bruker AMX 500 Spectrometer (500 MHz). All chemicals 2',4',2,4,-Tetrahydroxychalcone (2): C H O , colorless
15 12 6

for tyrosinase inhibition assay, including mushroom amorphous powder. ESI MS (m/z) : 271 [M-H]−. H- 1

tyrosinase, L-tyrosine, and kojic acid, were purchased NMR (500 MHz, CD OD): δ 8.09 (1H, d, J = 15.4 Hz, H-
3

from Sigma-Aldrich Chimical Co.. β), 7.88 (1H, d, J = 8.9 Hz, H-6), 7.70 (1H, d, J = 15.4
Plant Material − The cortex of Morus bombycis was Hz, H-α), 7.51 (1H, d, J = 8.4 Hz, H-6'), 6.39 (1H, dd,
collected in Nambu forest of Seoul National University, J = 2.3, 8.9 Hz, H-5), 6.36 (1H, dd, J = 2.2, 8,4 Hz, H-5'),
Baegwoon Mountain, Gwangyang city, Jeollanamdo, 6.34 (1H, d, J = 2.2 Hz, H-3'), 6.27 (1H, d, J = 2.3 Hz, H-
Korea, in September 2008. A voucher specimen (SNU- 3). C-NMR (125 MHz, CD OD): δ 194.9 (C = O),
13
3

0785) has been deposited in the Herbarium of the 168.1 (C-2), 166.9 (C-4), 163.6 (C-4'), 161.6 (C-2'), 142.9
Medicinal Plant Garden, College of Pharmacy, Seoul (C-β), 133.8 (C-6), 133.1 (C-6'), 118.4 (C-α), 116.4 (C-
National University. 1'), 115.6 (C-1), 109.9 (C-5'), 109.8 (C-5), 104.6 (C-3),
Extraction and Isolation − The air-dried cortex of 104.3 (C-3').
Morus bombycis (4.5 kg) was extracted with 80% MeOH Oxyresveratrol (3): C H O , colorless amorphous
15 12 6

in an ultrasonic apparatus at room temperature and 329.3 powder. ESI MS (m/z) : 243 [M-H]−. H-NMR (300 1

g of total extract was obtained. This extract was suspended MHz, CD OD): δ 7.37 (1H, d, J = 9.1 Hz, H-6), 7.20 (1H,
3

in distilled water and successively partitioned with n- d, J = 16.4 Hz, H-7), 6.74 (1H, d, J = 16.4 Hz, H-7'), 6.40
Hexane, EtOAc, and n-BuOH. Silica gel column chroma- (2H, d, J = 2.1 Hz, H-2', 6'), 6.23 (1H, d, J = 2.3 Hz, H-3),
tography (CC) of EtOAc fraction (78.2 g) was carried out 6.23 (1H, dd, J = 7.6, 2.3 Hz, H-5), 6.04 (1H, t, J = 2.0
using CHCl followed by a mixture of CHCl -MeOH in a
3 3 Hz, H-4'). C-NMR (75 MHz, CD OD): δ 160.2 (C-3'),
13
3

gradient system, and yielded nine subfractions (E1 ~ E9). 159.9 (C-4), 158 (C-2), 142.9 (C-1'), 129.1 (C-6), 127.2
Fraction E4 was subjected to silica gel CC (CHCl - (C-7'), 125.5 (C-7), 118.6 (C-1), 109.9 (C-5), 106.4 (C-2',
MeOH, 50 : 1 → 1 : 1) to give 10 subfrations (E4-1 ~
3

6'), 104.3 (C-3), 103.0 (C-4').

E4-10). Fraction E4-7 was also separated by MPLC using Moracinoside M (4): C H O , whitish amorphous
24 26 9

MeOH-H O gradient (30% - 90% MeOH) elution to afford

2 powder. ESI MS (m/z) : 453 [M-H]− , 325 [M - Xyl - H]−.
compound 1 (20.0 mg), and other subfrations. Fraction 1
H-NMR (400 MHz, CD OD): δ 7.22 (1H, s, H-4), 6.99
3

E4-7-8 was purified over HPLC (55% MeOH) to yield (1H, s, H-4'), 6.86 (1H, s, H-7), 6.49 (1H, s, H-2'), 3.79
27.6mg of compound 2. Fraction E5 was separated by (1H, dd, J = 7.4, 5.4 Hz, H-2''), 3.13 (1H, d, J = 16.4, 5.2
MPLC using MeOH-H O gradient (20% - 80% MeOH)
2 Hz, H-1''a), 2.84 (1H, dd, J = 16.4, 7.5 Hz, H-1''b), 1.35
elution to give 20 subfractions (E5-1 ~ E5-20). Fraction (3H, s, H-4''), 1.27 (3H, s, H-5''), xylopyranoside δ 4.89
E5-4 was purified over HPLC (70% MeOH) to yield (1H, d, J = 7.1 Hz, xyl-H1), 3.97 (1H, dd, J = 11.3, 5.2
258.8 mg of compound 3. Fraction E6 was separated by Hz, xyl-H5β), 3.59 (1H, br m, xyl-H4), 3.3 - 3.46 (2H, m,
MPLC using MeOH-H O gradient (30% - 90% MeOH)
2 xyl-H2 and xyl-H3), 3.36 (1H, br s, xyl-H5α). C-NMR 13

elution to afford 19 subfractions (E6-1 ~ E6-19). Fraction (100 MHz, DMSO-d6): δ 161.2 (C-5'), 160.7 (C-3'), 156.9
E6-14 was purified using HPLC (60% MeOH) to yield (C-2), 156.8 (C-7a), 153.5 (C-6), 134.5 (C-1'), 124.9 (C-
30.2 mg of compound 4. Fraction E7 was separated by 3a), 122.7 (C-4), 118.6 (C-5), 107.4 (C-6'), 106.2 (C-4'),
MPLC using MeOH-H O gradient (30% - 80% MeOH)
2 105.9 (C-2'), 103.1 (C-3), 100.5 (C-7), 79 (C-3''), 71.3 (C-
elution and yielded 17.7 mg of compound 5. 2''), 33.2 (C-1''), 26.8 (C-5''), 21.8 (C-4''), xylopyranoside
7,2',4'-Trihydroxyflavanone (1): C H O colorless
15 12 4, δ 103.7 (xyl-C1), 78.5 (xyl-C2), 75.5 (xyl-C3), 71.8 (xyl-
amorphous powder. ESI MS (m/z) : 271 [M-H]−. H- 1
C4), 67.7 (xyl-C5).
NMR (500 MHz, acetone-d6): δ 7.70 (1H, d, J = 8.6 Hz, Moracin M-3'-O-β-D-glucopyranoside (5): C H O , 20 20 9

H-5), 7.29 (1H, d, J = 8.4 Hz, H-6'), 6.53 (1H, dd, J = 8.6, brown amorphous powder. ESI MS (m/z) : 405 [M + H] . +
200 Natural Product Sciences

1
H-NMR (400 MHz, DMSO-d6) : δ 7.17 (1H, s, H-3),
7.40 (1H, d, J = 8.4 Hz, H-4), 6.75 (1H, dd, J = 8.4, 2.0
Hz, H-5), 6.94 (1H, br s, H-7), 6.97 (1H, br s, H-2'), 6.44
(1H, br s, H-4'), 6.89 (1H, br s, H-6'), glucopyranisode δ
4.63 (1H, d, J = 7.4 Hz, glc-H1), 3.20 - 3.24 (1H, m, glc-
H2), 3.27 - 3.34 (1H, m, glc-H3), 3.20 - 3.24 (1H, m, glc-
H4), 3.27 - 3.34 (1H, m, glc-H5), 3.53 (1H, dd, J = 11.7,
5.8 Hz, glc-H6), 3.75 (1H, dd, J = 11.1, 3.7 Hz, glc-H6).
13
C-NMR (100 MHz, DMSO-d6) : δ 159.0 (C-3'), 158.7
(C-5'), 155.8 (C-6), 155.3 (C-7a), 153.5 (C-2), 131.7 (C-
1'), 121.2 (C-4), 120.7 (C-3a), 112.5 (C-5), 104.7 (C-6'), Fig. Tyrosinase inhibitory effects of total extract and its
1.

103.6 (C-2'), 103.3 (C-4'), 102.1 (C-3), 97.5 (C-7), fractions of Morus bombycis. The concentration of the treated
glucopyranisode δ 101.0 (glc-C1), 73.3 (glc-C2), 76.6 samples was 100 µg/ml. *P < 0.05, significantly different from
the blank reference.
(glc-C3), 69.7 (glc-C4), 77.1 (glc-C5), 60.7 (glc-C6).
Tyrosinase Inhibitory Assay − The total extract, Table 1. Mushroom tyrosinase inhibitory activity of compounds
fractions, and isolated compounds were dissolved in 1 - 5

DMSO, and then diluted to several concentration in 0.1 M Compounds IC50 (µM) SDa
pH 6.8 sodium phosphate buffer. DMSO was added to
make the DMSO concentration of each sample equally 1 5.228 1.079
(final conc. 0.1%). The sample solutions (70 µL) were set 2 0.9724 1.073
on the 96-well plate, followed by the addition of 30 µL of 3 3.66 1.122
mushroom tyrosinase solution (1000 units/mL) and 40 µL 4 61.3 1.087
of 3 mM L-tyrosine solution. 70 µL of 0.1% DMSO- 5 127.5 1.285
buffer solution and 70 µL of kojic acid solution were used Kojic acidb 112.7 1.178
as the blank reference and positive control, respectively. a
SD : Standard deviation. IC50 value is calculated by logC-%inhi-
The absorbance at 490 nm of the reaction mixture bition plot, so SD value is the antilog of standard error of logIC50.
These values mean geometric SD, or a number that you can mul-
(140 µL) was measured using the ELISA reader, and the tiply the mean by or divide the mean into.
mixture was incubated for 10 min at 37 C. After the
o b
Kojic acid : Positive control.
incubation, the absorbance of mixture was measured
again. The inhibition percentage of tyrosinase activity was compounds were identified as 7,2',4'-trihydroxyflavanone
calculated as follows: (1) (Lim et al., 2001), 2',4',2,4,-tetrahydroxychalcone (2)
%inhibition = (A2 − A1) − (B2 − B1) / (A2 − A1) × 100 (Lim et al., 2001), oxyreveratrol (2',4',3,5-tetrahydroxy-
stilbene) (3) (Kanchanapoom et al., 2002), moracinoside
A1 is the absorbance at 490 nm of the blank reference M (4) (Jeong et al., 2009), and moracin M-3'-O-β-D-
at 0 min, and A2 is the absorbance at 490 nm of the blank glucopyranoside (5) (Kanchanapoom et al., 2002) by
at 10 min. B1 is the absorbance at 490 nm of the test comparing the H-, C-NMR and MS spectral data with
1 13

sample at 0 min, and B2 is the absorbance at 490 nm of the literature values.

the test sample at 10 min. Compounds 1 - 5 were tested for their mushroom
tyrosinase inhibitory effect (Table 1). Compounds 1, 2,
Results and Discussion and 3 exhibited strong inhibitory activities on tyrosinase
with IC values of 5.23 ± 1.08, 0.97 ± 1.07, and 3.66 ±
50

For the isolation of bio-active compounds from Morus 1.12, respectively, which were more potent than that of
bombycis cortex, the inhibitory effects of total extract and positive control, kojic acid. In addition, The inhibitory
the fractions (including n-Hexane, EtOAc, BuOH and effect of compound 2 was shown to be more potent than
water fractions) of this plant on tyrosinase were evaluated. that of compound 3 (oxyresveratrol) reported previously
As shown in Fig. 1, the total extract showed a potent as a major ingredient that contributes to skin whitening
tyrosinase inhibitory activity, and the EtOAc fraction was effect of Morus plants (Shin et al., 1998). To exhibit their
the most potent among the four solvent fractions. EtOAc mechanism of inhibition, we performed kinetic study for
fraction was further subjected to repeated column compounds 1 and 2. As a result, both compounds 1 and 2
chromatography yielded five pure compounds. The isolated were shown to play as competitive inhibitors on
Vol. 17, No. 3, 2011 201

control, kojic acid. Nerya et al. (2004) demonstrated that

inhibitory activities of phenolic compounds might be due
to the structural similarity to tyrosine. They also
demonstrated that 2,4-resorcinol functional group perform
a critical role in the tyrosinase inhibitory activity of
chalcones (Katib et al. 2005). Compound 2 has these two
functional resorcinol units, so it showed powerful inhibitory
potency. The isolated compounds herein are thought to
contribute at least in part to skin-whitening effect of
Morus bombycis.

Fig. 2. The structures of - isolated from M. bombycis.

1 5
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compound 3 (Jeong et al., 2009) (Fig. 3 and Fig. 4). In
addition, compound 4 and 5 also effectively attenuated Received May 12, 2011
Revised August 12, 2011
tyrosinase activity which was comparable to positive Accepted August 15, 2011