An efficient preparation of mulberroside a from the branch bark of mulberry and its effect on the inhibition of tyrosinase activity

Article abstract of DOI:10.1371/journal.pone.0109396

A bioactive ingredient in an ethanol extract from the branch bark of cultivated mulberry Husang-32 (Morus multicaulis Perr.) was isolated using a macroporous resin column. The primary component, which was purified by semi-preparative highperformance liqui

Full text of DOI:10.1371/journal.pone.0109396

An Efficient Preparation of Mulberroside A from the
Branch Bark of Mulberry and Its Effect on the Inhibition
of Tyrosinase Activity
1
1
2
1
Shu Wang , Xian-Ming Liu , Jian Zhang *, Yu-Qing Zhang *
1
Silk Biotechnology Laboratory, School of Basic Medical and Biological Sciences, Soochow University, Dushuhu Higher Edu. Town, Suzhou, P R China, 2 College of
Pharmaceutical Sciences, Soochow University, Dushuhu Higher Edu. Town, Suzhou, P R China
Abstract
A bioactive ingredient in an ethanol extract from the branch bark of cultivated mulberry Husang-32 (Morus multicaulis Perr.)
was isolated using a macroporous resin column. The primary component, which was purified by semi-preparative high-
performance liquid chromatography diode array detection (HPLC-DAD), was identified as mulberroside A (MA) by liquid
1
13
chromatograph-mass spectrometer (LC-MS), H and C nuclear magnetic resonance (NMR) spectra. In total, 4.12 g MA was
efficiently extracted from one kilogram of mulberry bark. The enzymatic analysis showed that MA inhibited the generation
of dopachrome by affecting the activities of monophenolase and diphenolase of tyrosinase in vitro. This analysis indicated
that MA and oxyresveratrol (OR), which is the the aglycone of mulberroside A, exhibited strong inhibition of the
monophenolase activity with IC50 values of 1.29 mmol/L and 0.12 mmol/L, respectively. However, the former showed weaker
inhibitory activity than the latter for diphenolase. For the monophenolase activity, the inhibitory activity of MA and OR was
reversible and showed mixed type 1 inhibition. Additionally, the inhibition constant KI (the inhibition constant of the
effectors on tyrosinase) values were 0.385 mmol/L and 0.926 mmol/L, respectively, and the KIS (the inhibition constants of
the enzyme-substrate complex) values were 0.177 mmol/L and 0.662 mmol/L, respectively. However, MA showed
competitive inhibition of diphenolase activity, and K
and K
I
was 4.36 mmol/L. In contrast, OR showed noncompetitive inhibition
= KIS = 2.95 mmol/L. Taken together, these results provide important information concerning the inhibitory
mechanism of MA on melanin synthesis, which is widely used in whitening cosmetics.
I
Citation: Wang S, Liu X-M, Zhang J, Zhang Y-Q (2014) An Efficient Preparation of Mulberroside A from the Branch Bark of Mulberry and Its Effect on the Inhibition
of Tyrosinase Activity. PLoS ONE 9(10): e109396. doi:10.1371/journal.pone.0109396
Editor: Thumballi R. Ganapathi, Bhabha Atomic Research Centre, India
Received June 24, 2014; Accepted September 3, 2014; Published October 9, 2014
Copyright: ß 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: The authors gratefully acknowledge the earmarked fund (CARS-22-ZJ0504) for China Agriculture Research System (CARS) and a project funded by the
Priority Academic Program Development of Jiangsu Higher Education Institutions, P. R. China. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
*
Email: jianzhang@suda.edu.cn (JZ); sericult@suda.edu.cn (YQZ)
Introduction
China, Korea, Japan and so on.). In fact, early in 2002 the Chinese
Ministry of Health declared that the mulberry leaf and mulberry
Tyrosinase, which is a polyphenol oxidase, is the key enzyme in
melanin synthesis and possesses monophenolase and diphenolase
activity. This enzyme can catalyse two distinct reactions: the
oxidation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA)
(fruit) were not only food, but also drugs; and the root bark (Cortex
Mori albae) and branch were health food especially the root was
widely used in the folk prescription of traditional Chinese
medicine. Modern pharmacological study proves that the aqueous
or methanol extracts of Cortex Mori have anti-inflammatory,
hypoglycemic, hypotensive, anticarcinogenic and anti-microbial
activities; and the chloroform and alkali extracts have the obvious
anti-inflammation and anti-asthmatic activities.
(
(
first reaction) and the oxidation of L-DOPA to dopaquinone
second reaction). Then, dopaquinone, through a non-enzyme-
catalysed process, is transformed into leukodopachrome (third
reaction). Leukodopachrome is oxidised into dopachrome by a
dopaquinone (fourth reaction), which is an extremely fast and non-
enzyme-catalysed process. Then, dopachrome is transformed to
melanin through a series of chemical- and enzyme-catalysed
reactions. The process of tyrosinase catalysing L-tyrosine to
dopachrome shows that dopachrome synthesis can be suppressed
when any of the steps are inhibited. Additionally, not all
substances that can inhibit the formation of dopachrome are
tyrosinase inhibitors, for example, thymol [1].
Mulberroside A (MA) is an oxyresveratrol diglycopyranoside,
where 4-OH and 39-OH are substituted by two glucose molecules
(Fig. 1). MA has cis and trans configurations. Early in 1986, this
compound was first isolated from the acetone extract of Morus
lhou Koidz root. After gavaging with aqueous extracts of mulberry
root, Qiu et al. found that the stilbenes absorbed by rats were
mostly MA [2,3]. MA could also be separated from the alcohol
extract of Cortex mori [4], and 1.346% MA was obtained from the
root using ultrasonic extraction [5]. The tyrosinase inhibitory
activity of MA from the root of Morus albae was greatly enhanced
Mulberry (Morus) trees, belongs to the family of Moraceae,
have been widely cultivated in several sericulture countries (e.g.
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
by the bioconversion process [6]. In addition, the ethyl acetate
fraction of the alcohol extract from leaves [7] and the monomer
separated from twigs [8], 2-oxyresverstrol, also had strong
tyrosinase inhibitory activity. The deglucosylated form of MA
had more potent tyrosinase inhibitory activity than its glycosylated
form [9], and the IC50 value decreased from 42.06 mmol/L to
Materials and Methods
2
.1 Plant material
Fresh one-year-old branches of cultivated mulberry (Husang
No. 32) from Morus multicaulis Perr. were collected from the
mulberry field of Soochow University in November 2010.
1
5.15 mmol/L. Kim [10] et al. determined the kinetics and
mechanism for the inhibition of mushroom tyrosinase, which was
reversibly inhibited by oxyresveratrol (OR) as a non-competitive
inhibitor, with L-tyrosine as the substrate. Another study showed
that the tyrosinase inhibitory activity of OR (0.49 mmol/L) was
approximately 110-fold higher than that of MA (53.6 mmol/L),
which was isolated from the ethanol extract of the roots of Morus
alba, and the kinetics showed MA to be a competitive inhibitor of
mushroom tyrosinase, with L-tyrosine and L-DOPA as substrates
2.2 Preparation of the mulberry branch bark ethanol
extract
The ethanol extract of mulberry branch bark was prepared by a
previously described method [19], with slight modifications. The
bark was peeled from the mulberry branches, then air-dried and
milled. The bark powder was repeatedly extracted with an 80%
ethanol solution under reflux 3 times, and the insoluble material
was removed by filtration and by centrifugation. The resulting
extracts were combined and concentrated under reduced pressure
to obtain a residue and lyophilised into powders.
[6]. A mixture of OR and MA separated from the alcohol extract of
twigs showed strong inhibition of melanin synthesis [6,11].
Moreover, a decade later, a related medicinal analysis revealed
that MA, which was extracted from the mulberry root, had
antitussive, anti-asthmatic [12], expectorant [13], anti-diabetic [14],
cell protection [15,16], anti-oxidative, hepatoprotection [17,18] and
other pharmacological effects.
2.3 Isolation on a macroporous resin column
A weighed amount of the branch bark powder was dispersed in
water, added to a D101 macroporous resin chromatography
column (Shanghai Blue Season Science and Technology Devel-
opment Co., Ltd.) and eluted with a gradient elution of ethanol-
The above-mentioned research concerning the separation,
purification, and identification of MA and its tyrosinase inhibition
were primarily identified from the mulberry root. There is only
rare systematic research and analyses regarding the extraction and
separation of mulberry branches, which are often used as
agricultural waste or as firewood. Thus far, there is a lack of
systemically studies and analyses concerning the mechanism of the
strong inhibitory action of MA on the monophenolase and
diphenolase activities of the mushroom tyrosinase. In this study,
we extracted an alcohol solution from Ramulus mori of the
cultivated mulberry (Husang No. 32) Morus multicaulis Perr.
Using a macroporous resin column for adsorption and for
separation and then purification by reversed-phase high-perfor-
mance liquid chromatography (RP-HPLC), we obtained much
MA monomer. Next, we systemically studied and analysed the
mechanism of the inhibition activity of MA on the mushroom
tyrosinase.
2
water (EtOH-H O, 0%, 30%, and 100%). The fractions were
concentrated and lyophilised into powders.
2.4 RP-HPLC for analysis and preparation
The experimental conditions of RP-HPLC for analysis and
semi-preparation were as follows:
A Shimadzu LC-20A HPLC pump connected to a Shimadzu
VP-ODS column (25064.6 mm) was used to analyse the samples.
All the sample used was precisely formulated into a solution of
0
.5 mg/ml. Mobile phases A (0.4% aqueous acetic acid) and B
acetonitrile) were prepared according to the following procedure:
(
initial 0-20 min, linear change from A/B = 95:5 (v/v) to A/
B = 85:15 (v/v); 20–30 min, linear change from A/B = 85:15 (v/v)
to A/B = 80:20 (v/v); 30–35 min, linear change from A/B = 80:20
(v/v) to A/B = 45:55 (v/v) and then held for 5 min. The mobile
phase flow-rate was set at 1.0 mL/min, and aliquots of 10 ml were
injected for analysis. The detection wavelength was 324 nm,
scanning from 200 nm to 600 nm. All analyses were performed at
room temperature.
The preparation of MA standard curve was as follows. In total,
1
0 mg of MA standard was dissolved in water and then formulated
into a solution of 1.0 mg/mL. Samples of 2.0, 4.0, 6.0, 8.0, 10.0,
2.0, and 14.0 mL of MA standard solution were injected into the
1
HPLC system. A standard curve was drawn, with the peak area as
the vertical axis (y) and with the standard injection volume (mg) as
the abscissa (x).
The sample preparation was performed using a Semi-Prepar-
ative HPLC (Shimadzu LC-6A) equipped with a DAD detector
(SPDM20A). The semi-preparative column consisted of a Shim-
pack Prep-ODS (H) column (250620 mm) fitted with a guard
module (Shim-pack G-ODS). Mobile phase A was 0.4% aqueous
acetic acid, and mobile phase B was acetonitrile (Sinopharm
Chemical Reagent Co., Ltd). The detection wavelength was
3
24 nm, scanning from 200 nm to 600 nm. All preparations were
performed at room temperature. The gradient program used was
as follows: 0–30 min, linear change from A/B = 93:7 (v/v) to A/
B = 80:20 (v/v); 30–40 min, linear change from A/B = 80:20 (v/v)
to A/B = 50:50 (v/v) and then held for 10 min. The flow rate was
Figure 1. Structure of mulberroside A. (Oxyresveratrol-4-O-b-D-
glucopyranosyl-39-O-b-D-glucopyranoside or 2, 4, 39, 59-tetrahydroxys-
tilbene).
5
.0 mL/min, and aliquots of 5.0 mL were injected. The peak
corresponding compound was collected at the retention time of
33.148 min and then concentrated and lyophilised into powders.
doi:10.1371/journal.pone.0109396.g001
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
2.5 LC-MS analysis
The liquid chromatograph-mass spectrometer (LC-MS) condi-
according to the following linear procedure: 0–10 min, A/
B = 90:10 (v/v).
tions were as follows: a Q-Trap 2000 LC/MS/MS electro-spray
ionisation source (ESI source) from ABI Company; an Agilent 1100
LC system; a Shimadzu Shim-Pack VP-ODS (250;mm62.0 mm)
chromatographic column, a column temperature of 18uC; mobile
phase: 10% acetic acid aqueous solution:acetonitrile = 60:40 (v/v);
2.8 Determination of the tyrosinase inhibition
mechanism
2.8.1 The inhibition of the tyrosinase monophenolase and
diphenolase activity. The measurement of the sample’s
inhibitory activity on monophenolase activity was based on
0.5 mmol/L L-tyrosine as the substrate [20]. The total reaction
volume was 3.0 mL, which included 2.8 mL substrate solution,
100 mL different concentrations of the sample solutions and
100 mL mushroom tyrosinase solution. The substrate and enzyme
(+)
Enhanced Mass Spectrometry (EMS ) scanning, 380uC; DP: 25;
CUR: 30; G1: 40; G2: 35; EP: 10; CE: 10.
2.6 NMR analysis
The lyophilized powder of the sample was dissolved using
.5 mL deuterated methanol (CD OD) and 0.5 mL deuterated
0
dimethyl sulphoxide (CD SOCD ), respectively. The carbon
3
2 4 2 4
solution were dissolved in 50 mmol/L NaH PO -Na HPO
temporary buffer solution (pH 6.8), with a substrate concentration
of 0.5 mmol/L and with an enzyme solution concentration of
1.0 mg/mL. The samples were dissolved in DMSO. First, 100 mL
sample solution was added, then 2.8 mL substrate solution, which
was pre-incubated at 30uC, was added, and last, 100 mL enzyme
solution was added and immediately mixed. The curve was drawn
using real-time tracking of the optical density change over time at
3
3
1
3
1
spectrum ( C-NMR) and hydrogen spectrum ( H-NMR) were
measured using a 300 MH superconductive nuclear magnetic
resonance (NMR) spectrometer.
Z
2
.7 Determination of tyrosinase activity inhibition
2
.7.1 The effect on the total process of dopachrome
4
75 nm. The measuring instrument was a Hitachi U3000 UV-
synthesis. The effect of tyrosinase (Worthington, USA) on the
catalysis of L-tyrosine to dopachrome was determined using the
method described by Hiroki [1], with slight modifications. The
total reaction volume was 3 mL. First, 2.8 mL of 2.0 mM L-
tyrosine was incubated at 30uC for 5 min, and then 100 mL of
visible spectrophotometer. The enzyme activity was obtained from
the slope of the line. The sample concentration-response curve was
plotted as the inhibition rate of samples to the enzyme against the
concentrations of effectors. The IC50 value, which represented the
concentration of sample at which 50% of the tyrosinase activity
was inhibited, was obtained from the curve.
1
.0 mM sample solution and 100 mL of 2.0 mg/mL enzyme
solution were added and mixed. The change in the absorption
spectrum was determined using a Hitachi U3000 UV-visible
spectrophotometer at 30uC isothermal conditions. The scanning
wavelength ranged from 250 nm to 600 nm. The scanning
frequency was 2 min, and the scanning time was 12 min. Thus,
the changes in the absorption spectrum over time were obtained.
The measurement of the sample’s inhibitory activity on
diphenolase activity was based on 0.5 mmol/L L-DOPA as the
substrate. The experimental procedure and calculations were
identical to those above-mentioned procedures and calculations.
2.8.2 The inhibition mechanism of the tyrosinase
monophenolase or diphenolase activity. The concentration
2
.7.2 The effect of the enzyme-catalysed process on
dopachrome synthesis. The effect of the enzyme-catalysed
process was determined using a previously described method [1],
with slight modifications. In total, 100 mg N-acetyl-L-tyrosine was
dissolved using NaH PO -Na HPO temporary buffer solution
of the substrate was fixed in 3.0 mL of 50 mmol/L NaH
Na HPO temporary buffer solution (pH 6.8); however, the
2 4
PO -
2
4
amount of enzyme was changed to determine the effects of
different concentrations of sample on the reaction of mushroom
tyrosinase catalysing L-tyrosine or L-DOPA as substrates.
Mapping the reaction rate in terms of the amount of enzyme
determined whether the inhibition was a reversible reaction. If the
lines all passed through the origin, then reversible inhibition was
demonstrated. However, if the lines were parallel, then irreversible
inhibition was demonstrated.
2
4
2
4
(pH 6.8), and the volume was set to 100 mL. In total, 0.25 mg/
mL enzyme solution and a certain concentration of the sample
solution were prepared. All the solutions were filtered using a
syringe filter with an aperture of 0.22 mm. The components of the
reaction system were as follows: 80 mL N-acetyl-L-tyrosine was
mixed with 10 mL sample solution and incubated at 30uC for
5
min, and then 10 mL enzyme solution was added. During the
And the inhibition type and inhibition constant were deter-
mined as follows. The concentration of the enzyme solution was
period from 0–60 min, 20 mL mixed solution was injected into the
HPLC system every 10 min to detect changes in N-acetyl-L-
tyrosine. The HPLC conditions were as follows: a Shimadzu
HPLC pump equipped with a Shimadzu 9082652 VP-ODS
column (250 mm64.6 mm). The detection wavelength was
2 4 2 4
fixed in 3.0 mL of 50 mmol/L NaH PO -Na HPO temporary
buffer solution (pH 6.8); however, the amount of substrate (L-
tyrosine or L-DOPA) was changed to determine the effects of
different concentrations of effectors on the reaction of mushroom
tyrosinase catalysing the substrate. Using the Lineweaver-Burk
method [6], the curve was plotted as the reciprocal of the reaction
rate 1/V against the reciprocal of the substrate 1/[S] to determine
the type of inhibition. If the lines intersected on the y-axis, then
competitive inhibition was indicated. If the lines intersected on the
x-axis, then noncompetitive inhibition was indicated. If the lines
intersected in the first or fourth quadrant, then this inhibition was
mixed type 1 or 2. If the lines were parallel, then anticompetitive
inhibition was indicated. The inhibition constant of the effectors
2
75 nm. The injection volume was 10 mL, and the flow-rate was
1
.0 mL/min. The mobile phases were A (water) and B (acetoni-
trile), which were prepared according to the following linear
procedure: 0–8 min, A/B = 85:15 (v/v). All analyses were
performed at room temperature.
2.7.3 The effect of the non-enzyme-catalysed process on
dopachrome synthesis. The effect of the non-enzyme-cata-
lysed process was determined by a previously described method
[1], with slight modifications. The HPLC conditions were as
follows: a Shimadzu HPLC pump equipped with a Shimadzu VP-
ODS column (250 mm64.6 mm). The detection wavelengths
were 245 nm and 280 nm. The injection volume was 20 mL, and
the flow-rate was 1.0 mL/min. The mobile phases were A (0.4%
aqueous acetic acid) and B (acetonitrile), which were prepared
I
on tyrosinase (K ) and the inhibition constants of the enzyme-
substrate complex (KIS) were obtained by a secondary plot of the
concentration of the effectors against the slope of the line and the
y-axis intercept, respectively.
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
Results
chromatogram (Fig. 3B), also showed that a single peak was only
obtained at the retention time of 21.05 min. This monomer was
used in the following NMR analysis.
3.1 The preparation of Mulberroside A
In total, 2.0 kg powder as treated using the previously described
1
13
method. Finally, 350.68 g ethanol extract was obtained. The
content of the ethanol extract was 17.5% of the powder. Then, the
ethanol extract was dissolved in water at 2%,5% concentrations
and loaded onto a column packed with D101 macroporous resin.
Following D101 macroporous resin column chromatography, the
ethanol extract was divided into three parts: 259.19 g for the water
elution fraction (W), 76.59 g for the 30% ethanol elution fraction
3
.4 LC-MS, NMR ( H, C spectrum) Identification
The compound obtained was identified using MS and NMR.
MS spectra showed the following positive ion ESI-MS m/z
+
+
+
values: 591.2 [M+Na] , 569.2 [M+H] , 406.9 [M-162] , 245.0
+
[
M-162-162+H] , and showed that the molecular weight of the
compound was 568.2.
1
3
H-NMR (CD OD, 300 MHz) d: 7.34 (1H, d, J = 9.3 Hz, H-6),
(
E30) and 5.65 g for E100. The results suggested that the bark
ethanol extract was primarily composed of W (73.91%) and E30
21.84%) and that the volume of E100 was small (1.61%). This
7
6
.22 (1H, d, J = 16.5 Hz, a-H), 6.84 (1H, d, J = 16.5 Hz, b-H),
.68 (1H, brs, H-69), 6.54 (1H, m, H-5), 6.53 (1H, m, H-29), 6.49
1H, m, H-3), 6.36 (1H, brs, H-49), 4.79 (1H, d, J = 7.5 Hz, C-
(glc)H-10), 4.79 (1H, d, J = 7.5 Hz,C-39 (glc)H-190), and 3.85–
.21 (12H, m). Anomeric H signals in the 3.85–3.21 (12H, m) are
(
(
HPLC analysis showed that the composition of E30 was relatively
simple; therefore, subsequent experiments were focused on E30.
4
3
overlapping signals. The original NMR spectrum has been added
as Figure S1.
13
3.2 Isolation and Purification
Using RP-HPLC to analyse E30, the analysis of the 3D
3
C-NMR (CD OD, 75 MHz) d: 120.2 (C-1), 157.1 (C-2),
chromatogram (Fig. 2(A)) demonstrated that the composition of
E30 was relatively simple, and at the retention time of 21.52 min,
E30 had a single strong peak corresponded to the main ingredient
of E30. In the 3D chromatogram, the maximum absorption peak
of the main ingredient was at 324 nm. To have a clearer view of
the separation efficiency, we transferred the 2D chromatogram at
the 324 nm wavelength from the 3D chromatogram (Fig. 2(B),
blue line). As shown in Fig. 2(B), the retention time of 21.52 min
showed an extremely sharp peak. The separation efficiency was
excellent and suitable for use in the semi-preparative HPLC for
further separation and for purification. The red line shown in
Fig. 2(B) denotes the chromatogram of the main ingredient of E30
using the semi-preparative HPLC. The fraction of the peak was
collected, concentrated and lyophilised to obtain the purified
compound, which had a total weight of 8.24 g.
1
1
1
04.8 (C-3), 160.4 (C-4), 109.3 (C-5), 128.4 (C-6), 127.6 (b-C),
24.9 (a-C), 141.9 (C-19), 107.2 (C-29), 159.5 (C-39), 103.9 (C-49),
59.5 (C-59), 108.1 (C-69), 102.0 (C-4(glc)C-10), 74.8 (C-20), 77.9
(C-30), 71.3 (C-40), 78.1 (C-50), 62.4 (C-60), 102.3 (C-39 (glc)C-190),
7
4.9 (C-290), 77.9 (C-390), 71.3 (C-490), 78.1 (C-590), and 62.5 (C-
90). The original NMR spectrum has been added as Figure S2.
6
1
H-NMR (CD SOCD , 400 MHz) d: 9.80 (1H, s, OH), 9.38
3
3
(1H, s, OH),7.44 (1H, d, J = 8.4 Hz, H-6), 7.22 (1H, d,
J = 16.4 Hz, a-H), 6.94 (1H, d, J = 16.4 Hz, b-H), 6.64 (1H, brs,
H-69), 6.58 (1H, m, H-29), 6.52 (1H, d, J = 8.4 Hz, H-5), 6.55 (1H,
m, H-3), 6.35 (1H, brs, H-49), 4.79 (1H, d, J = 7.2 Hz, C-4(glc)H-
1
0), 4.79 (1H, d, J = 7.2 Hz,C-39 (glc)H-190), 5.24 (2H, dd,
J = 10.0 Hz, 4.4 Hz), 5.10 (4H, m), 4.51 (2H, d, J = 6.4 Hz),
.70 (2H, m), 3.50 (3H, m), and 3.18–3.30 (7H, m). Both anomeric
3
H signals in the 3.50 (3H, m) and 3.18–3.30 (7H, m) are
overlapping signals. The original NMR spectrum has been added
as Figure S3.
3.3 RP-HPLC-DAD analysis
There were no other interfering peaks, except at the retention
1
13
1
By comparing the H-NMR (CD OD), C-NMR and H-
3
9, 16
time of 21.05 min in the 3D chromatogram (Fig. 3A), which
showed that the compound obtained by semi-preparative HPLC
was of high purity. Transferring the 200–500 nm UV-visible scan
spectra from the 3D chromatogram (Fig. 3C) showed that the
compound had a maximum absorption value at 324 nm. The 2D
chromatogram at 324 nm, which was transferred from the 3D
NMR (CD SOCD ) data with the literature
3
, the compound
3
was identified as oxyresveratrol-4-O-b-D-glucopyranosyl-39-O-b-
D-glucopyranoside or mulberroside A (MA). Its molecular formula
is C H O , and the structure is shown in Fig. 1.
2
6
32 14
Figure 2. 3D (A) and 2D profiles (B) of E30 fraction on analytic and prepared HPLC-DAD.
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
Figure 3. 3D(A),2D(B) profiles on HPLC and UV-visible spectra(C)of MA.
doi:10.1371/journal.pone.0109396.g003
3
.5 The inhibition of MA on dopachrome synthesis
When no inhibitor was added, tyrosinase continuously catalysed
have been previously studied; however, there have only been a few
studies concerning MA. Whether MA and OR can influence the
third reaction of dopachrome synthesis has not been reported, and
our next experiment was designed to determine whether this
influence occurred.
L-tyrosine to dopaquinone and to dopachrome during the period
from 0–12 min (spectral scanning every 2 min); thus, the contents
of dopaquinone and dopachrome in the reaction system rose
(Fig. 4a). Additionally, the results are shown for when 2.0 mmol/L
MA and 2.0 mmol/L OR were added (Fig. 4b and Fig.4c).
Compared with no inhibitor added, dopaquinone and dopa-
chrome also increased with time; however, the rate slowed down,
which indicated that MA and OR inhibited the formation of
dopachrome. The effects of OR on the inhibition of tyrosinase
3.6 The effect MA on tyrosinase activity (monophenolase
and diphenolase activity)
N-acetyl-L-tyrosine was used instead of L-tyrosine as the
substrate for mushroom tyrosinase (Fig. 5) because the mono-
phenolase activity of the mushroom tyrosinase can catalyse the
Figure 4. Consecutive UV-Vis spectra obtained in the oxidation of 0.5 mmol/L L-tyrosine by mushroom tyrosinase in the absence
sample (a), in presence of 2 mmol/L mulberroside A (b) or oxyresveratrol (c) for 12 min. Spectra scans were repeatedly at a speed of
300 nm/30 s in 2 min intervals.
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
Figure 5. Using N-acetyl-L-tyrosine as a substrate, because of the presence of an acetate group on the N-group of an amino acid,
intracyclization of dopaquinone to leukodopachrome is blocked.
doi:10.1371/journal.pone.0109396.g005
conversion of N-acetyl-L-tyrosine to N-acetyl-L-DOPA and
because diphenolase can catalyse N-acetyl-L-DOPA to N-acetyl-
dopaquinone. Due to an acetate group on the amino of N-acetyl-
dopaquinone, cyclisation cannot occur; thus, the reaction was
blocked. Therefore, changes in N-acetyl-L-tyrosine in the reaction
system can directly reflect the influence of the sample on
mushroom tyrosinase activity. If the sample added to the reaction
system cannot inhibit the activity of mushroom tyrosinase, then
the content of N-acetyl-L-tyrosine will gradually decrease over
time (the peak height of N-acetyl-L-tyrosine that can be observed
Figure 6. The change in the content of N-acetyl-L-tyrosine as a substrate for tyrosinase in the absence of sample (A), in the
presence of 175.9 mmol/L MA (B) or oxyresveratrol (C) during 60 min. HPLC analysis of the redox reaction of p-benzoquinone and
L-DOPA in the absence of sample (D), or in the presence of mulberroside A (E), or in the presence of oxyresveratrol (F) at 245 nm.
HPLC profiles of the redox reaction of p-benzoquinone and L-DOPA in the absence of PBS (G), mulberroside A (H) or oxyresveratrol
(I) at different reaction times of 0, 10, 20 and 30 min.
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
Figure 7. Inhibition effects of mulberroside A (blue line) and oxyresveratrol (red line) on the monophenolase activity of mushroom
tyrosinase.
doi:10.1371/journal.pone.0109396.g007
in the HPLC chromatogram will decrease); however, if the activity
of mushroom tyrosinase is inhibited, then the content of N-acetyl-
L-tyrosine will not change over time (the peak height of N-acetyl-
L-tyrosine will not change or will decrease extremely slowly in the
HPLC chromatogram).
tyrosinase in the reaction system. However, when added to
175.9 mmol/L MA and OR, the content of N-acetyl-L-tyrosine
did not change over the period from 0–60 min. These results
showed that MA and OR inhibited the activity of tyrosinase,
resulting in no change in N-acetyl-L-tyrosine content throughout
the reaction.
Fig. 6A, B, and C show the changes of N-acetyl-L-tyrosine
content in the reaction system when N-acetyl-L-tyrosine, as a
substrate, was added to the buffer solution, to 175.9 mmol/L MA,
and to 175.9 mmol/L OR, respectively. The retention time of N-
acetyl-L-tyrosine in the column was 6.5 min. Without inhibitors,
the figures show that N-acetyl-L-tyrosine decreased by 44.35%
after 20 min, by 60.53% at 40 min, and by 73.07% after 1 h.
These results showed that N-acetyl-L-tyrosine was transformed by
3.7 The inhibition of MA on dopaquinone to
dopachrome
To measure whether the samples affected the second stage of
the dopachrome synthesis, we used L-DOPA and p-benzoquinone
to substitute leukodopachrome and dopaquinone to simulate the
non-enzymatic process. L-DOPA and p-benzoquinone were
Figure 8. The reversible inhibitory reaction of Mulberroside A (Left) and oxyresveratrol (Right) on monophenolase of mushroom
tyrosinase.
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
Figure 9. Determination of the inhibitory type and inhibition constant of MA (Left) and oxyresveratrol (Right) on monophenolase
of mushroom tyrosinase. (A) Lineweaver-Burk plots for inhibition of MA or oxyresveratrol on monophenolase. (B) and (C)represent the plots of
slope and intercept versus the concentration of MA for determining the inhibition constant KI and KIS.
doi:10.1371/journal.pone.0109396.g009
converted to dopaquinone and to hydroquinone by a redox
reaction. HPLC was used to detect the changes in p-benzoquinone
and hydroquinone contents over time in the reaction system, and
the results are shown in Fig. 6D-F. The maximum absorption
peaks of p-benzoquinone and hydroquinone were at 245 nm and
Fig. 6, in a certain concentration range (0,3.5 mmol/L), the
inhibition rate increased with increased concentrations of the
sample. However, there were differences in the inhibitory activity
between the two substances. MA and OR resulted in similar
inhibition. The IC50 values of MA and OR were 1.29 mmol/L and
0.12 mmol/L, respectively. These results indicated that MA and
OR strongly inhibited the monophenolase activity of the
mushroom tyrosinase. The inhibitory activity of OR was
approximately 10-fold stronger than that of MA, which indicated
that the two glucose molecules added to OR had some effect on
the inhibition of the monophenolase activity of the mushroom
tyrosinase.
2
4
80 nm, respectively, and the retention times were 7.76 min and
.86 min in the Shimadzu VP-ODS column (250 mm64.6 mm),
respectively. Fig. 6D–F shows the HPLC chromatogram at
45 nm wavelength, and Fig. 6D shows the change in p-
benzoquinone when no samples were added to the system. Within
0 min, p-benzoquinone decreased by 75%, and at 20 min, p-
2
1
benzoquinone decreased by 90.62%. Fig. 6E and F represent the
results when MA and OR were added. The results indicated that
the declining trend in p-benzoquinone content did not change and
was not suppressed by the addition of MA and OR. As shown in
Fig. 5G–I, the content of hydroquinone also did not change at
3.9 The inhibition mechanism of MA on the
monophenolase activity of tyrosinase
By adding different concentrations of samples to the reaction
system, when the concentration of L-tyrosine was unchanged, but
the amount of enzyme added was changed, the reaction velocity
2
80 nm when compared with the analysis when the samples were
not added. These results showed that the redox reactions of L-
DOPA and p-benzoquinone were not influenced by MA and OR.
Therefore, MA and OR do not play a role in the conversion of
dopaquinone to dopachrome, which further illustrates that MA is
a real tyrosinase inhibitor, similar to OR, and has inhibitory
activity on both monophenolase and diphenolase.
(V) of the enzyme-catalysed oxidation of the substrate was
measured by the change in the amount of enzyme ([E]). OR
was the control in this test, which measured the mechanism by
which MA inhibits the monophenolase activity of tyrosinase. The
selected sample concentrations of MA were 0, 0.22 mmol/L,
0
.44 mmol/L, 0.88 mmol/L and 1.76 mmol/L; the sample
3.8 The inhibition of MA on monophenolase activity of
tyrosinase
The samples that inhibited the monophenolase activity of
tyrosinase were measured using L-tyrosine as the substrate. In the
reaction system, OR was included for comparison. Fig. 7 shows
the effects of different concentrations of MA on the inhibition of
the monophenolase activity of mushroom tyrosinase. As shown in
concentrations of OR were 0, 0.21 mmol/L, 0.42 mmol/L,
0.84 mmol/L and 1.68 mmol/L; and the enzyme concentrations
were 0.8 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL and
2.5 mg/mL. The results are shown in Fig. 8. A group of straight
lines through the origin was obtained for both MA and OR in the
system, and the slope of the line decreased with increased
concentrations of the samples, which indicated that the inhibitory
Table 1. Inhibition effect of mulberroside A and of oxyresveratrol on the monophenolase activity of the mushroom tyrosinase.
Sample
IC50 (mmol/L)
Inhibition mechanism
Inhibition type
K
I
(mmol/L)
KIS (mmol/L)
MA
OR
1.29
0.12
reversible
reversible
mix-type I
mix-type I
0.385
0.926
0.177
0.662
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
second quadrant. This result illustrated that increased sample
concentrations not only led to a decrease in the maximum reaction
velocity (Vmax) of the enzymatic reaction but also to an increase in
the Michaelis-Menten constant (K ). These results showed that
m
MA inhibited the monophenolase of the mushroom tyrosinase by
a mixed-type inhibitory pattern, which was identical to OR. The
I
inhibition constants of MA and OR on free enzyme (K ) and on
the enzyme-substrate complex (KIS) were obtained by plotting the
slope and the y-axis intercept against the sample concentration
(Table 1), respectively.
3.11 The inhibition of MA on the diphenolase activity of
tyrosinase
The samples that inhibited the diphenolase activity of tyrosinase
were measured with L-DOPA as the substrate. Different
concentrations of MA or OR were added to the reaction system
to monitor their effects on the diphenolase activity of the
mushroom tyrosinase. The results are shown in Fig. 10. In a
certain concentration range, in which the inhibition rate increased
with increased sample concentrations. However, there were
differences between MA and OR on the inhibition of diphenolase
activity. The IC50 values of MA and OR were 7.45 mmol/L and
0.39 mmol/L, respectively. These results showed that OR had the
strongest activity on diphenolase, whereas MA was weaker.
Compared with MA, the IC50 value of OR was 19-fold lower.
This result indicated that the inhibition of diphenolase was
weakened after the two glucose molecules were added to OR.
Figure 10. Inhibition effects of mulberroside A (blue) and
oxyresveratrol (red) on the diphenolase activity.
doi:10.1371/journal.pone.0109396.g010
effect of MA and OR on the monophenolase of the mushroom
tyrosinase was a reversible process. In addition, the effects of MA
and OR on the activity of the enzyme were caused by the
inhibition of enzyme activity rather than irreversible inactivation.
Therefore, both MA and OR were reversible inhibitors of the
mushroom tyrosinase.
3.12 The inhibition mechanism of MA on the tyrosinase
diphenolase
The concentration of the substrate, L-DOPA, was unchanged in
the reaction system; however, the amount of enzyme added was
changed. The relation between the velocity of the enzyme-
catalysed oxidation of the substrate (V) and the amount of enzyme
3.10 The inhibition constant of MA on the
monophenolase activity of tyrosinase
The concentration of enzyme in the reaction system was
constant. The impact of different concentrations of samples on
enzyme activity was measured by changing the concentration of L-
tyrosine and using Lineweaver-Burk double reciprocal plots. The
reciprocal of the substrate concentration 1/[S] was the x-axis, and
the reciprocal of the reaction rate 1/V was the y-axis. The results
are shown in Fig. 9. A group of straight lines intersected in the
([E]) was measured after adding different sample concentrations.
The concentrations of MA were 0, 2.0 mmol/L, 4.0 mmol/L and
8.0 mmol/L; the concentrations of OR were 0, 0.1 mmol/L,
0.2 mmol/L and 0.4 mmol/L; and the enzyme concentrations were
0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL and 1.0 mg/mL. A plot of
absorbance change against the amount of enzyme (Fig. 11)
showed that both MA and OR resulted in a group of straight
Figure 11. The inhibitory mechanism of Mulberroside A (a) and oxyresveratrol (b) on diphenolase of mushroom tyrosinase was
reversible.
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
Figure 12. Inhibition constant of MA (a) and oxyresveratrol (b) on diphenolase of mushroom tyrosinase. (A) Lineweaver-Burk plots for
inhibition of MA on diphenolase. (B) The plots of slope versus the concentration of mulberroside A for determining the inhibition constants KI,
respectively.
doi:10.1371/journal.pone.0109396.g012
lines through the origin, and the slope of the lines decreased with
increased sample concentrations. These results illustrated that the
inhibition mechanisms of MA and of OR on the diphenolase
activity was a reversible process and that both MA and OR
inhibited the diphenolase activity but did not lead to irreversible
enzyme inactivation. Therefore, these experimental results further
showed that both MA and OR were reversible inhibitors of the
mushroom tyrosinase. However, the differences in IC50 values
between MA and OR showed that OR was the stronger reversible
inhibitor, whereas MA was the weaker inhibitor.
line against sample concentration were obtained to determine the
inhibition constants of MA on the free enzyme (K = 4.36 mmol/L)
I
and OR on the enzyme-substrate complex (KIS = K
L) (Table 2).
= 2.95 mmol/
I
Discussion
Tyrosinase is a polyphenol oxidase and is not only the key
enzyme in the food browning reaction [21] but is also involved in
mammalian melanin formation [22]. Enzymatic browning in fresh
fruits and vegetables usually leads to the loss of nutrition [23].
Thus, the use of tyrosinase inhibitors appears to be a promising
approach to suppress undesirable browning reactions to help
maintain the quality of food products and can be used as an
important additive in whitening products in the cosmetics
industry. Well-known examples include arbutin, kojic acid and
glabridin. These compounds have good inhibitory effects; howev-
er, their uses are limited and, therefore, remain not widely used.
Therefore, the identification of a tyrosinase inhibitor that has a
wide range of sources, a good inhibitory effect and no adverse
effects has become a focus of research in recent years.
3.13 The inhibition constant of MA on the diphenolase
activity of tyrosinase
The concentration of the enzyme in the reaction system was
constant. The impact of different concentrations of samples on the
enzyme activity was measured by changing the concentration of L-
6
DOPA and using Lineweaver-Burk double reciprocal plots . The
reciprocal of the substrate concentration 1/[S] was the x-axis, and
the reciprocal of the reaction rate 1/V was the y-axis. The results
are shown in Fig. 12. The double reciprocal plots of MA and of
OR showed significantly different results. A group of lines that
intersected on the y-axis was obtained in the plot of MA, which
indicated that the maximum reaction velocity (Vmax) did not
change when the sample concentration increased; however, the
Concerning the molecular structure analysis, the hydrogen of
the two phenolic hydroxyls at the 49 and 39 position in OR were
substituted by two glucose molecules, which slightly affected the
activity of MA in inhibiting the monophenolase activity of
tyrosinase, and the IC50 values only had 10-fold differences.
However, both of these hydroxyls had strong inhibition that is a
mixed inhibition type. However, the presence of the two glucose
molecules had a significant effect on the inhibition of the
diphenolase activity of the tyrosinase. The IC50 value of MA
was only 7.45 mmol/L and was almost 20 times that of OR. The
inhibition types of MA and OR on the diphenolase activity were
Michaelis-Menten constant (K ) increased. This result indicated
m
typical competitive inhibition. The lines obtained by the double
reciprocal plot of OR intersected on the x-axis, which indicated
that the Michaelis-Menten constant (K ) was not affected by
m
increased sample concentrations but led to decreased maximum
reaction velocity (Vmax). This result indicated non-competitive
inhibition. Secondary plots of the slope and y-axis intercept of each
Table 2. Inhibition effects of MA and of OR on the diphenolase activity of the mushroom tyrosinase.
Sample
IC50 (mmol/L)
Inhibition mechanism
Inhibition type
K
I
(mmol/L)
KIS (mmol/L)
MA
OR
7.45
0.39
reversible
reversible
competitive
4.36
2.95
—
Non-competitive
2.95
—: none.
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The Preparation of Mulberroside A and Inhibitory Effect on Tyrosinase
different. These findings illustrated that the presence of the two
glucose molecules seriously affected the diphenolase inhibitory
activity.
side from waste mulberry branches in the sericulture industry
using the method reported in this article. The extract also showed
strong tyrosinase inhibitory activity. Therefore, this method can be
used in the reclamation of MA from large mulberry biological
resources, which can not only be widely used in whitening
cosmetics but also have important development potential in anti-
oxidative and anti-aging biological drugs, as well as in health
foods.
Our results were both similar and different when compared with
the previous findings reported by others. The results concerning
the effect of OR on the inhibition of the mushroom tyrosinase
were similar to our results. The strong tyrosinase inhibitory activity
of OR was previously confirmed in other studies, which found that
the IC50 value of OR was 0.46,1.10 mmol/L, depending on the
assay conditions [8,24] Concerning the OR-inhibited monopheno-
lase activity, the IC50 value of OR extracted from the mulberry
twigs was 0.943 mmol/L (0.23 mg/L), which was reported by Lee et
al. [8]. Compared with our results, there was an 8-fold difference in
the IC50 value. The IC50 values of OR, which inhibited the
monophenolase and diphenolase activities, were approximately 4-
fold and 30-fold lower than that of our experiment, respectively.
Their inhibition mechanism and type were also identical [6].
However, the IC50 value of MA for the inhibition of the
monophenolase activity was almost 40-fold lower than our results,
and the inhibition type was different. Kim’s results showed
competitive inhibition, whereas our results showed mixed-type
inhibition. In addition, our results showed that MA had apparent
inhibition of the diphenolase, which differed from their reports [24].
These inconsistent results were most likely caused by differences in
the purity of the sample, the source of the mushroom tyrosinase, the
reaction systems and operator errors. We continue to thoroughly
examine these issues.
MA has a strong inhibitory activity on tyrosinase. However,
compared with OR, which has a similar structure, there are few
reports concerning the biological activity and function of MA. MA
also has poor permeability [24]. Nevertheless, a pharmacokinetic
analysis in 1996 showed that a small number of MA derivatives
were found in the blood of rats after oral administration of the
mulberry root acetone extract, and the bioavailability was only
approximately 1%. This result was because most of the MA was
transformed to OR and transferred into the circulating blood, and
the absorption rate was estimated to be 50%. OR was also rapidly
transported into the tissues [3]. Three major metabolites were
isolated from rat faeces, including oxyresveratrol-2-O-b-D-gluco-
side, oxyresveratrol-39-O-b-D-glucoside and OR. This result
indicated that MA was metabolised before being absorbed [25].
Following incubation with intestinal bacteria under anaerobic
conditions, MA was rapidly deglucosylated and transformed into
OR or generated aglycon-oxyresveratrol [26]. OR has anti-
bacterial, anti-inflammatory [26], and neuroprotective [27]
activities and can repair liver injury induced by alcohol [28].
Therefore, MA used as an oral preparation can be converting into
OR in vivo. This conversion into OR can increase its permeability
and then can easily be absorbed by cells, which can use a variety of
physiological activities of OR. Thus, the pharmacological effects of
MA should be studied together with OR to obtain in-depth
knowledge concerning these compounds.
Conclusions
In this study, only D101 macroporous resin and a semi-
preparative HPLC system were used to efficiently extract MA
from mulberry twigs. The purity of MA was as high as 98.3%. The
enzymatic analysis showed that MA and OR had strong inhibitory
activities on the mushroom tyrosinase. In vitro, MA inhibited the
synthesis of dopachrome primarily by inhibiting the two steps of
the first process, which inhibited tyrosinase activity, and had
weaker inhibitory activity on diphenolase. However, MA had no
effect on the non-enzymatic process.
Tyrosinase inhibitory activity analyses showed that MA resulted
in strong inhibition of the monophenolase activity of the
mushroom tyrosinase. Additionally, the IC50 value was
1
.29 mmol/L, which had a significant difference compared with
the inhibitory activity of OR (IC50: 0.12 mmol/L). Compared with
OR (IC : 0.39 mmol/L), MA showed weaker inhibition of the
50
diphenolase of the mushroom tyrosinase, and the IC50 value was
7.45 mmol/L. The analyses of the inhibition mechanism of the
monophenolase and diphenolase of the tyrosinase indicated that
MA showed reversible inhibition, which was similar to OR. The
inhibition activities of MA and of OR on the monophenolase of
tyrosine was mixed-type 1. Additionally, the inhibition constants
K were 0.385 mmol/L and 0.926 mmol/L, respectively, and K
I
IS
values were 0.177 mmol/L and 0.662 mmol/L, respectively. For
diphenolase activity, there were some differences between MA and
OR. MA showed competitive inhibition, and K
L; however, OR showed noncompetitive inhibition, and K
IS = 2.95 mmol/L.
was 4.36 mmol/
I
I
= -
K
Supporting Information
Figure S1 1H-NMR (CD3OD, 300 MHz) spectrum of the
MA.
(PDF)
Figure S2 13C-NMR (CD3OD, 75 MHz) spectrum of the
MA.
(PDF)
Figure S3 1H-NMR (CD3SOCD3, 400 MHz) spectrum of
the MA.
(PDF)
Although Wu [5] et al. obtained 1.346% MA using ultrasonic
extraction, the MA was extracted from the root, and the method
was extremely complicated. From the analysis of our experiments,
we can efficiently extract 0.412% of oxyresveratrol diglycopyrano-
Author Contributions
Conceived and designed the experiments: YQZ. Performed the experi-
ments: SW XML. Analyzed the data: SW XML. Contributed reagents/
materials/analysis tools: SW XML. Wrote the paper: SW XML.
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October 2014 | Volume 9 | Issue 10 | e109396