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Enhancement of Tyrosinase Inhibitionof the Extract of Veratrum patulumUsing Cellulase
Dong Hyun Kim,1,2 Jin Hee Kim,1 Seung Hwa Baek,1 Jin Ho Seo,2
Yung Hee Kho,1 Tae Kwang Oh,1 Choong Hwan Lee1
1Korea Research Institute of Bioscience and Biotechnology,Daejon 305-333, Korea; telephone: 82 42 860-4294;fax: 82 42 860-4595; e-mail: [email protected] of Agricultural Biotechnology, Seoul National University,Seoul 151-742, Korea
Received 18 December 2003; accepted 8 April 2004
Published online 18 August 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20189
Abstract: Inhibitors of melanin biosynthesis were screenedby using three different methods. The extract of Veratrumpatulum contains hydroxystilbene compounds that arepotent tyrosinase inhibitors. We evaluated the enzyme in-hibitory property on the mushroom tyrosinase of hydroxy-stilbene compounds including resveratrol, oxyresveratrol,and their analogs. Biotransformation using cellulase of thewhole extract brought about an increase in the inhibitoryactivity of the products on mushroom tyrosinase. The en-hancement of tyrosinase inhibition is supposed to increasethe concentration of aglycon, which has superior inhibitoryactivity to its glycoside. Eventually, melanin biosynthesiswas inhibited by the enhanced tyrosinase inhibitory activityof the extract. This result indicated that deglycosylation ofstilbene compounds has exerted more effective inhibitionon the enzyme than that of the unprocessed plant extract.B 2004 Wiley Periodicals, Inc.
Keywords: biotransformation; cellulase; tyrosinase inhib-itor; resveratrol; Veratrum patulum
INTRODUCTION
A widely distributed plant polyphenol oxidase, tyrosinase is
of central importance in vertebrate pigmentation and in the
browning of fruits and vegetables. This enzyme catalyzes
the o-hydroxylation of monophenols using molecular oxy-
gen (monophenolase activity), and the oxidation of o-
diphenols to o-quinones (diphenolase activity). Tyrosinase
converts tyrosine into DOPA and then converts DOPA into
DOPA-quinone (Rodriguez-Lopez et al., 1992; Sanchez-
Ferrer et al., 1995; Solomon et al., 1996). DOPA-quinone
produced by tyrosinase is nonenzymatically converted to
dopachrome, which is acted upon by an isomerase pro-
ducing dihydroxyindoles (April et al., 1998; Kuriyama et al.,
1998). Melanin pigments are eventually produced by further
oxidation and polymerization of the indoles (Riley, 1997).
Because of its central role in melanogenesis, tyrosinase has
become a key target of screening required in the discovery of
new inhibitors.
Plant polyphenols have been the targets of several studies,
as a result, their classification, occurrence, structural aspects,
reactivity, biochemistry, and biogenesis have been repeat-
edly reviewed (Haslam et al., 1989). Polyphenols is a broad
term used in the literature on natural products for defining
substances that possess one or more benzene ring(s) and
hydroxyl groups, including functional derivatives. They
constitute one of the most abundant and ubiquitous groups of
plant metabolites (Ho et al., 1992). Moreover, dietary poly-
phenols are recognized for their beneficial implications for
human health, and polyphenols are usually found in various
type and extent of chemical substitution in plants (Bravo,
1998; Ursini and Sevanian, 2002). Much literature is avail-
able on the screening of tyrosinase inhibitors among phe-
nolics of plant origin, and polyphenols are currently the
target of intense studies (Nakayama et al., 2001). When these
phenolic compounds show a good affinity for tyrosinase,
dopachrome formation is prevented. However, the affinity
on the tyrosinase could be interfered with by the chemical
substitution of the compounds.
The hydrolytic enzymes were produced by microorga-
nisms for the hydrolysis of polysaccharides to metaboliz-
able products (Warren, 1996). The inorganic and organic
chemistry communities have been actively involved in
developing models and mimics of hydrolytic enzymes. Their
diverse function, for example, glycoside hydrolytic activities
with different specificities and mode of action, would be
required for efficient modification of natural compounds and
plant extract.
We now present a screening of potent tyrosinase inhibitors
and the tyrosinase inhibitory effect of hydroxystilbene
compounds. Also we describe the enhancement of tyrosinase
inhibition using enzymes and the selection of the appropriate
enzyme for the enzymatic reaction for the production of
B 2004 Wiley Periodicals, Inc.
Correspondence to: C.H. Lee
Contract grant sponsor: Ministry of Science and Technology, Korea
Contract grant number: PF0321205-00, MG02-0401-001-1-0-0
tyrosinase inhibitor. The melanin contents in melanocytes
were quantified with biotransformed plant extract.
MATERIALS AND METHODS
Materials
Mushroom tyrosinase, L-tyrosine, and L-DOPA were
purchased from Sigma Chemical Co. (St. Louis, MO).
The oxyresveratrol and related hydroxystilbene analogs
were isolated from various herbal extracts or obtained by
chemical modifications (Ryu et al., 1988). The methanol
extracts of various plants were obtained from Plant Extract
Bank, KRIBB (Daejon, Korea).
Assay of Tyrosinase Inhibition
The DOPA and tyrosine oxidase activities of mushroom
tyrosinase were spectrophotometrically determined as
described previously with minor modifications (Matsuda
et al., 1994). Sixty AL of 3 mM L-DOPA or 1.5 mM L-
tyrosine, 80 AL of 100 mM phosphate buffer (pH 6.8) and
60 AL of the same oxybuffer with or without test sample
were added to a 96-well microplate (Nunc, Denmark), and
then 20 AL of mushroom tyrosinase (500 U/mL) was
mixed. After incubating at 37jC for 20 min, the amount of
dopachrome produced in the reaction mixture was deter-
mined as the optical density at 490 nm (OD 490) by using a
PowerWave X340 microplate reader (BIO-TEK Instrument
Inc., USA). The inhibitory effects on the enzyme activity
by the test samples were represented as % of inhibition,
1 � SampleOD490
Control OD490
� �� 100:
The data were collected as the mean F standard error (n = 3),
and their significance was analyzed by the Student’s t-test.
IC50 represents the concentration of the sample, which
inhibits 50% of the enzyme activity.
Assay of Inhibitory Effect of Melanin Productionin Streptomyces bikiniensis
Streptomyces bikiniensis was used for estimating the
melanogenetic inhibition zone. Melanin synthesis inhib-
itory activity was determined by the paper-disc agar
diffusion method. A preserved culture of S. bikiniensis
NRRL B-1049 was inoculated on a Papavizas’ VDYA agar
slant, which contained V-8 juice (Campbell Soup Co.)
200 mL, glucose 2 g, yeast extract (Difco) 2 g, CaCO3 1 g,
agar (Difco) 20 g and distilled water 800 mL, the pH ad-
justed to 7.2 before autoclaving. After incubation at 28jC
for 2 weeks, 2 mL of sterile water was added onto the slant
culture, and the spore mass formed on the aerial mycelium
was scraped with an inoculating loop. The spore suspension
thus obtained was transferred to sterile micro tubes. 0.4 mL
of the spore suspension of S. bikiniensis was added to the
agar medium ISP No. 7 (40 mL) supplemented with Bacto-
yeast extract (Difco) 0.2% and was spread over the agar
surface uniformly with a glass hockey bar. After the agar
surface was dried, a paper disc (8-mm diameter) soaked
with sample solution was placed on the agar plate. The
plate was incubated at 28jC for 48 h; the diameter zone
(mm i.d.) of melanin formation was measured from the
reverse side of the plate.
Melanization Inhibition Assay on Melan-a Cell
Cells were seeded into a 24-well plate (Falcon, Lincoln
Park, NJ) at a density of 1 � 105 cells per well and al-
lowed to attach overnight. The medium was replaced with
fresh medium containing various concentrations of com-
pounds. Cells were cultured for 72 h and further incubated
for a day. After washing them with phosphate-buffered
saline (PBS), the cells were lysed with 250 AL of 0.85 N
KOH and transferred to a 96-well plate. The melanin
contents were estimated by measuring the absorbance at
405 nm. Phenylthiourea (PTU) was used as a positive control
(Bennett et al., 1987).
Western Blot Analysis
Cells were grown in a 6-well plate and treated with
enzymatic reaction mixtures. They were washed with ice-
cold PBS 3 times and lysed in cold lysis buffer (0.1M Tris-
HCl, pH 7.2, 1% Nonidet P-40, 0.01% SDS, 1 mM
phenylmethylsufonyl fluoride, 10 Ag/mL leupeptin, 1 Ag/
mL aprotinin). An aliquot of lysate was used to determine the
protein concentration by the Bradford method (#500-0002,
Bio-Rad, Hercules, CA). Thirty micrograms of protein per
lane was separated by 8% SDS-polyacrylamide gel electro-
phoresis. The resolved proteins were transferred to a PVDF
membrane (Millipore, Bedford, MA) at 250 mA for 2 h. The
membranes were blocked with 5% skim milk for 1 h and
washed with 0.05% TBST (TBS containing 0.05% Tween
20). The membranes were then incubated for 2 h with the
antibody for tyrosinase. Tyrosinase and h-actin were de-
tected by using the rabbit polycolonal anti-aPEP7 anti-
body (1:1000, a gift from Dr. V.J. Hearing, National
Institutes of Health, Bethesda, MD) and the mouse mono-
colonal anti-h-actin antibody (1:5000, Sigma, St, Louis,
MO). The tyrosinase and h-actin were then further incubated
with horseradish peroxidase-conjugated secondary anti-
body. Bound antibodies were detected by using the
Amersham ECL system. The expression of h-actin was
used as a normalizing control.
In Vitro Biotransformation
Biotransformation was performed under strictly anaerobic
conditions using an in vitro batch system. Two milliliters
of the enzyme solutions were added to each sealed
bioreactor containing 10 mg of plant extract suspended in
8 mL of appropriate buffer, and the headspace was rinsed
850 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 87, NO. 7, SEPTEMBER 30, 2004
with CO2 for 1 min. After equilibrating the internal
pressure, the vials were closed hermetically and incubated
in a shaking water bath (150 rpm) at 37jC. Each sample
and blank was used as reaction controls. All samples were
prepared in duplicate. The microbial activity was termi-
nated by addition of 0.1 mL of a saturated HgCl2 solution.
After the incubation, each aliquot was centrifuged at 4jC
for 10 min. To determine the substrate disappearance and
the metabolite production, 0.5 mL of the supernatant was
stored frozen until determined by HPLC.
HPLC Analysis of the Product After Reaction
After thawing, each sample was centrifuged at 9860g for
15 min, and the supernatant was blown with N2 gas until
dryness. The residue was reconstituted with 1 mL meth-
anol, and it was subjected to HPLC analysis.
The HPLC system consisted of a Hitachi Model L-7100
intelligent pump and an L-4000 UV detector. A CapcellPak
C18 (10 � 250 mm, Shiseido, Tokyo, Japan) column with a
guard column (MetaGuard 4.6 mm Polaris 5 m C18-A,
MetaChem, Torrance, CA) was employed. The detector
wavelength was set at 254 nm, and the flow rate was
1.5 mL/min. The mobile phases used for the analysis of
various samples were mixtures of 0.1% (v/v) phosphoric
acid (A) and methanol (B) with isocratic or gradient elu-
tion. The gradient elution was 20–100% B in 30 min at a
flow rate of 1.5 mL/min.
RESULTS
Tyrosinase Inhibitor Screening
Melanin is principally responsible for skin color and
plays an important role in the prevention of sunburned skin
injury. Biosynthesis of melanin which is produced by
melanocytes in the basal layer of epidermis, starts from
the conversion of the amino acid L-tyrosine to 3,4-dihy-
droxyphenylalanine (L-DOPA), and then the oxidation of
L-DOPA yields DOPA-quinone by tyrosinase, an enzyme
catalyzing the rate-limiting step for the melanin biosyn-
thesis (Sanchez-Ferrer et al., 1995). Therefore, tyrosinase
inhibitors have been established as important constituents
of cosmetic materials and depigmenting agents for
hyperpigmentation (Seo et al., 2003). In this regard, we
explored the tyrosinase inhibitor over 3,000 standardized
plant extracts at Plant Extract Bank in Korea. Table I
shows the inhibitory activities of various plant extracts on
mushroom tyrosinase. Tyrosinase inhibitors represent an
interference of melanin biosynthesis of S. bikiniensis and
decrease ultimately the melanin content in Melan-a cell.
Most good tyrosinase inhibitors in the extracts reduced the
production of melanin. The poorest tyrosinase inhibitor
in Table I, however, was the crude extract of Veratrum
patulum, which belongs in Liliaceae. Several plants in
Liliaceae have reportedly included bioactive components
known to be antifungal agents and tyrosinase inhibitors
(Ryu et al., 1988; Takahama and Hirota, 2000). The extract
seemed to contain some stilbene compounds and their
derivatives, which were identified as resveratrol and its
glycosides and methoxides with chromatographic methods
(data not shown). The extract of Veratrum patulum was ex-
pected to inhibit tyrosinase effectively since resveratrol has
been studied as an inhibitor of tyrosinase by many
investigators (Kim et al., 2002; Shin et al., 1998). Contrary
to the expectation, resveratrol and its derivatives in the
Veratrum patulum extract could not inhibit tyrosinase
effectively. We assumed, therefore, that there is a directly
proportional relationship between the contents of resvera-
trol-related compounds and the tyrosinase inhibitory activ-
ity. To evaluate the validity of this assumption, we measured
the inhibitory activity of tyrosinase by comparing the piceid,
a representative glycosylated stilbene, and its aglycon,
the resveratrol.
Tyrosinase Inhibitory Effect ofHydroxystilbene Compounds
More than 30 stilbenes and stilbene glycosides occur natu-
rally among the members of the plant kingdom classified as
spermatophytes. The essential structural skeleton comprises
two aromatic rings joined by an ethylene bridge. Table II
shows the inhibitory effects of hydroxystilbene compounds
from the extract of Veratrum patulum on the oxidation of
DOPA and tyrosine, major activities of mushroom tyrosin-
ase. Resveratrol exhibited potent inhibitory effect on the
DOPA and tyrosine oxidase of mushroom tyrosinase.
Significant inhibitions on oxidase activities of mushroom
tyrosinase shown by resveratrol suggested that it is a poten-
tial candidate of skin-whitening agents. Piceid is a glyco-
side of resveratrol at position 3, and rhaponticin is a
glycoside of rhapontigenin at position 3V. However, piceid
has 6.9- and 8.2-fold less activities for DOPA and tyro-
sine oxidation than that of resveratrol. None of the
glycosylated hydroxystilbene compounds showed signifi-
cant inhibitory effects at 100 AM on tyrosine oxidation
activity of mushroom tyrosinase. Therefore, the variety
Table I. Inhibitory activities of various plant extracts in melanin
biosynthesis.
Name
Tyrosinase
inhibition
IC50 (Ag/mL)
Melanogenetic
inhibition
zone (mm)
Melanin
content
(%)
Morus bombycis 1.0 40 28
Glycyrrhiza uralensis 3.5 25 36
Broussonetia kazinoki x papyrifera 5.0 29 49
Lespedeza cyrtobotrya 12.5 25 57
Vitis coignetiae 25.0 23 60
Salix floderusii 30.0 15 69
Broussonetia kazinoki var. humilis 50.0 20 99
Acer barbinerve 50.0 19 94
Lespedeza X robusta 50.0 10 103
Veratrum patulum 100.0 9 77
KIM ET AL.: ENHANCEMENT OF TYROSINASE INHIBITION OF THE EXTRACT OF VERATRUM PATULUM USING CELLULASE 851
of hydroxyl groups on phenyl rings of parent stilbene
skeleton seems to be helpful to its inhibitory effect on
tyrosinase. The results indicate that piceid, a glycoside of
hydroxystilbene, did not exhibit significant inhibition in
comparison with the parent hydroxystilbene, resveratrol.
Glycosylation and methylation of stilbene compounds do
not affect the oxidation activity of the enzyme. While the
resveratrol glycosides, methoxides, and polymers are the
most abundant stilbenes in nature, their relative skin-
whitening activity is generally negligible in comparison
with resveratrol and oxyresveratrol (Kubo and Kinst-Hori,
1999). These findings led us to suggest that the deglyco-
sylation of glycosylated hydroxystilbene compounds is
attributed to and intensified by an ability to inhibit tyro-
sinase activity. We introduce the application of hydro-
lyzing enzymes to deglycosylation for efficient hydrolysis
of glycosylated hydroxystilbene compounds.
Biotransformation of Veratrum patulum Extract
In the last decade of the 20th century, there was an
explosive interest in resveratrol; it promised miraculous
health benefits (Savouret and Quesne, 2002; Sovak, 2001).
The major dietary sources of stilbenes include grapes, wine,
soybeans, peanuts, and peanut products although hydroxy-
stilbenes are present as glucosides rather than aglycon. We
discovered recently that Veratrum patulum is also a fine
production source for resveratrol with the same inherent
defect. Since resveratrol is more active than its glucoside—
piceid on inhibition of tyrosinase—the biotransformation of
piceid to resveratrol is required for better whitening effects.
We have described a novel enzymatic approach to efficient
production of resveratrol from the extract of Veratrum
patulum containing piceid at the same time. Figure 1 shows
the fact that tyrosinase inhibitory activity was enhanced in
the extract of Veratrum patulum by using polysaccharide-
degrading enzymes. Among polysaccharide-degrading en-
zymes, cellulase and h-glucosidase were suitable to
enhance the inhibitory effects in the extract. We were able
to observe the changes in the ratio and the contents of
products in accordance with the various enzymes used
despite the same concentration of each different enzyme.
The ratios of resveratrol and piceid in the extract of Vera-
trum patulum were monitored with HPLC (data not shown).
The constituents of the biotransformation were identified in
Figure 1. Inhibitory effect enhancement of the extract of Veratrum
patulum using polysaccharide degrading enzymes. Abbreviations:h-Glu:h-
Glucosidase; Hem: Hemicellulase; Dex: Dextranse; Cel: Cellulase; a-Glu:
a-Glucosidase; Amy: Amylase.
Table II. Chemical structure and mushroom tyrosinase inhibitory effects of hydroxystilbene compounds.
Substituent DOPA oxidation Tyrosine oxidation
Compound R1 R2 R3 R4 R5 Inhibition (%) IC50 (AM) Inhibition (%) IC50 (AM)
Oxyresveratrol H OH OH OH OH 97 F 0.7 2.3 98 F 1.9 1.2
Resveratrol H H OH OH OH 78 F 1.4 123.3 67 F 5.6 43.5
3,5-Dihydroxy-4V-methoxystilbene H H OMe OH OH 63 F 1.9 187.9 54 F 6.3 86.8
3,4V-Dimethoxy-5-hydroxystilbene H H OMe OMe OH 54 F 4.5 398.3 13 F 2.1 >100
Trimethylresveratrol H H OMe OMe OMe 37 F 2.6 > 500 3 F 0.8 >100
Piceid H H OH OH OGlc 14 F 1.6 > 500 12 F 1.7 >100
Rhaponticin H OGlc OMe OH OH 3 F 3.4 > 500 8 F 2.5 >100
852 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 87, NO. 7, SEPTEMBER 30, 2004
comparison with the authentic samples. From the retention
times on the HPLC chromatogram, the relationship
between polarity and structures of the stilbene compounds
in the extract could be proposed and identified with ESI-
MS repeatedly. It explains how the ratio of resveratrol,
which provides the enhancement of inhibitory effects, came
to be raised in the action of the enzymes. Figure 2 shows
the changes in product concentration and tyrosinase
inhibition according to the reaction time. The concentration
of glycoside of oxyresveratrol and resveratrol was
decreased by the cellulase, while their aglycon con-
centrations and the tyrosinase inhibitory activity increased.
The concentrations of resveratrol and oxyresveratrol were
proportional to the inhibitory effect on mushroom tyrosin-
ase. We suggest that the increased tyrosinase inhibi-
tion activity interferes ultimately with the biosynthesis
of melanin. As shown in Figure 3, melanin contents in
Melan-a cell were measured and the expression of tyro-
sinase enzyme was observed. The extract of Veratrum
patulum treated with cellulase did not affect protein ex-
pression but decreased about 20% of melanin contents by
inhibiting tyrosinase, a key enzyme in the biosynthesis
of melanin.
DISCUSSION
Tyrosinase converts tyrosine to 3,4-dihydroxy-phenylala-
nine (L-DOPA), and it oxidizes L-DOPA to form DOPA-
quinone, which plays an important role in the process
of melanin biosynthesis (Rodriguez-Lopez et al., 1992;
Sanchez-Ferrer et al., 1995; Solomon et al., 1996). Inhib-
itory effects on tyrosinase by resveratrol and some related
hydroxystilbene analogs were helpful for medical treate-
ment of local hyperpigmentations such as melasma,
ephelide, and lentigo. Since hydroxystilbene has been more
active than its glucoside on inhibition of tyrosinase, bio-
transformation of stilbene glycoside to aglycon was tried by
Figure 3. Effects of enzymatic reaction mixtures on melanogenesis in
Melan-a cells. (A) Tyrosinase expression, and (B) melanin contents were
measured as described in Materials and Methods.
Figure 2. Biotransformation of piceid to resveratrol and cumulative inhibitory effect (Gray bar) on mushroom tyrosinase. (.) Oxyresveratrol-diglucoside,
(o) oxyresveratrol-monoglucoside, (z) resveratrol-diglucoside, (q) resveratrol-monoglucoside, (n) oxyresveratrol, (5) resveratol.
KIM ET AL.: ENHANCEMENT OF TYROSINASE INHIBITION OF THE EXTRACT OF VERATRUM PATULUM USING CELLULASE 853
using degrading enzymes. The polysaccharide-degrading
enzymes have been traditionally used for the process of
deglycosylation of products (Aristidou and Penttila, 2000).
In the biotransformation process, cellulase is superior to
other degrading enzymes for this objective. It can enhance
the biological properties of stilbene aglycon as well as the
tyrosinase inhibitiory activity. Cellulase is the best-known
hydrolyzing enzyme and has a wide spectrum of substrates
(Bayer et al., 1998). These characteristics seem to be at-
tributed to the same biotransforming reaction with var-
ious plant extracts as substrate. Because it does not require
much time and effort, the enzymatic reaction would en-
able us to gather a wider variety of plant extract libraries as
well as much greater opportunity of serendipity at screening
steps. A screening process using the simple enzymatic bio-
transformation would be more helpful in investigating the
unique and megascopic biological properties in a library
of wider variety of extracts than using a single original
plant extract. Resveratrol was a major product of the re-
action using the extract of Veratrum patulum by cellulase.
Siemann and Creasy (1992) reported on the presence of
trans-resveratrol in wine and drew attention to the fact
that it was also a constituent of oriental folk medicines
reputed to benefit persons afflicted by a wide range of dis-
orders. In the process of biotransformation, the quantity of
resveratrol was increased and that of substrate, piceid in
reaction time, was decreased. The whitening activity of
the reactant was increased by the quantity of resveratrol
and reaction time. It explains that the increase of aglycon
concentration allows more inhibitory effect on tyrosinase,
and that the overall ratio of aglycon against its glycoside
increases. Therefore, we may expect the tyrosinase inhibi-
tory activity to increase as much as the tyrosinase inhibi-
tory activity through the enzyme reaction even if only a
relatively small amount is used. Eventually, the melanin
biosynthesis was inhibited by tyrosinase inhibitory activity
enhanced by cellulase. We verified that the enzymatic
reaction is effective on the biotransformation of glycoside
to aglycon in the crude extract of Veratrum patulum. The
Veratrum patulum extract may also be used as possible re-
sources that can offer sufficient inhibitory activity on the
tyrosinase. Further studies of various plant extracts using
hydrolyzing enzymes are in progress. And the mode of
cellulase action on the plant extract is required for the better
understanding of microenvironmental mechanisms.
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