Enhancement of the tyrosinase inhibitory activity of Mori Cortex Radicis extract by biotransformation using Leuconostoc paramesenteroides PR

Article abstract of DOI:10.1271/bbb.111002

Mori Cortex Radicis (MCR), the root bark of Morus alba L., consists of various phytochemicals and exhibits a strong inhibitory effect on tyrosinase. To enhance the tyrosinase inhibitory activity of MCR extract without further purification of bioactive compounds, whole MCR extract was biotransformed with crude enzyme extract from a selected lactic acid bacterium, Leuconostoc paramesenteroides PR (LP). Mulberroside A (MA), a major stilbene glucoside of MCR, contains two β-glucosyl residues at the C3 and C40 positions of oxyresveratrol (OXY). The crude enzyme of LP hydrolyzed the two glycosidic bonds of MA effectively, and 97.1% of MA was biotransformed into OXY within 2 h. Commercial almond β-glucosidase hydrolyzed only one site of the two glycosidic bonds of MA, and 68.7% of MA was biotransformed to OXY-glucoside. The tyrosinase inhibitory activity of the crude extract of MCR was increased approximately 6.5-fold by biotransformation using LP, and the IC₅₀ value of the transformed MCR was 3.7μg/mL.

Full text of DOI:10.1271/bbb.111002

Biosci. Biotechnol. Biochem., 76 (8), 1425–1430, 2012
Enhancement of the Tyrosinase Inhibitory Activity
of Mori Cortex Radicis Extract by Biotransformation
Using Leuconostoc paramesenteroides PR
y
1;2;
Jung Sung KIM,1 Hyun Ju YOU,1 Hye Yoon KANG,1 and Geun Eog JI
1Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National University,
Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
2Research Institute, BIFIDO Co., Ltd., 688-1 Sang-oan-ri, Hongchun-gun, Gangwon-do 250-804, Republic of Korea
Received January 6, 2012; Accepted May 16, 2012; Online Publication, August 7, 2012
[doi:10.1271/bbb.111002]
Mori Cortex Radicis (MCR), the root bark of Morus
alba L., consists of various phytochemicals and exhibits
a strong inhibitory effect on tyrosinase. To enhance the
tyrosinase inhibitory activity of MCR extract without
further purification of bioactive compounds, whole
MCR extract was biotransformed with crude enzyme
extract from a selected lactic acid bacterium, Leuco-
nostoc paramesenteroides PR (LP). Mulberroside A
(MA), a major stilbene glucoside of MCR, contains
two ꢀ-glucosyl residues at the C3 and C40 positions of
oxyresveratrol (OXY). The crude enzyme of LP hydro-
lyzed the two glycosidic bonds of MA effectively, and
97.1% of MA was biotransformed into OXY within 2 h.
Commercial almond ꢀ-glucosidase hydrolyzed only one
site of the two glycosidic bonds of MA, and 68.7% of
MA was biotransformed to OXY-glucoside. The tyrosi-
nase inhibitory activity of the crude extract of MCR was
increased approximately 6.5-fold by biotransformation
using LP, and the IC50 value of the transformed MCR
was 3.7 ꢀg/mL.
arisen because of the side effects of these compounds. In
this regard, various naturally occurring herbal extracts
and their active phytochemicals, such as flavonoid-like
agents and other derivatives, have gained attention as
potentially more desirable putative hypopigmenting
agents.4–6)
Botanical structures of the mulberry tree (Morus alba
L.), including the fruit, leaves, and root bark, have been
used widely for many years in traditional Oriental
medicine to treat fever, protect the liver, remove
sputum, improve eyesight, strengthen joints, facilitate
the discharge of urine, and lower blood pressure.7,8) The
root bark of Morus alba L., Mori Cortex Radicis (MCR),
is rich in phytochemicals such as 1-deoxynojirimycin,
ꢀ-aminobutyric acid, benzofuran derivatives, coumarins,
and various flavonoids including hydroxystilbene deriv-
atives.9,10) Stilbene glucosides such as mulberroside
A (MA), oxyresveratrol-2-O-ꢁ-D-glucopyranoside, and
oxyresveratrol-30-O-ꢁ-D-glucopyranoside comprise the
main components of MCR aqueous extract10,11) and are
not found in Morus alba L. leaves or fruits, suggesting
that there are significant differences in the components
of the root bark, leaves, and fruits.12)
Key words: biotransformation; tyrosinase inhibition;
Mori Cortex Radicis; Leuconostoc parame-
senteroides PR; oxyresveratrol
Currently, many studies are being conducted to
investigate the health benefits of MCR, including
antioxidative, antiviral, antiasthmatic, hypolipidemic,
antihyperglycemic, and antiatherogenic effects.13–18)
MA and its aglycone, oxyresveratrol (OXY), exhibit
antioxidant activities, and show an inhibitory effect
against FeSO4/H2O2-induced lipid peroxidation in rat
microsomes, and a scavenging effect on DPPH (1,1-
diphenyl-2-picrylhydrazyl) radicals.19,20) Oxidative
stress caused by free radicals has been proposed as a
contributing factor in hyperpigmentation, suggesting
that the antioxidant activity of MCR can serve as a
depigmenting agent. In addition, Lee et al. reported that
MCR extract exhibited the greatest tyrosinase inhibitory
effect among 285 oriental herb extracts and showed no
toxicity in eye irritation and skin sensitization tests on
animals, or in human skin irritation tests.21)
Tyrosinase (EC 1.14.18.1), also known as polyphenol
oxidase, is widely distributed in microorganisms, ani-
mals, and plants.1) It plays a significant role in the
modulation of melanin production, which is involved in
the formation of melanins, by catalyzing two distinct
reactions: the hydroxylation of L-tyrosine, and the
oxidation of L-DOPA (3,4-dihydroxyphenylalanine) to
DOPA-quinone.2) Hydroxylation by tyrosinase is the
first step, and a rate-limiting step of melanogenesis.
Many studies have focused on identifying an effective
tyrosinase inhibitor as a way to control tyrosinase
activity and consequent melanogenesis.3) Most reported
tyrosinase inhibitors are phenol/catechol derivatives
that have structures similar to substrates of tyrosinase,
such as tyrosine and DOPA.
Among reported tyrosinase inhibitors, hydroquinone,
arbutin, and kojic acid are known for their effectiveness
and have been used for many years in the cosmetics
industry as whitening agents, but safety concerns have
The content of MA in the MCR extract was 12-fold
higher than the content of OXY, but the biological
activities of OXY were much greater than those of MA.
In measurements of antioxidative activity against lipid
y
To whom correspondence should be addressed. Tel: +82-2-880-8749; Fax: +82-2-884-0305; E-mail: geji@snu.ac.kr
Abbreviations: LP, Leuconostoc paramesenteroides PR; MA, mulberroside A; MCR, Mori Cortex Radicis; OXY, oxyresveratrol

1426
J. S. KIM et al.
glucosidase from almond was used as positive control. After
incubation at 37 ꢁC for 30 min, the reaction was stopped by adding
100 mL of 1.0 M Na2CO3, and then the optical density (OD) was
measured at 405 nm (Model 680 Microplate Reader; Bio-Rad
Laboratories, Hercules, CA). At the same time, the protein concen-
tration of each enzyme extract was measured by the Bradford method.
One unit of activity defines as the amount of enzyme that releases
1 mmol of p-nitrophenol per min. Values are means of at least three
measurements. Relative activity against commercial ꢁ-glucosidase was
calculated by a comparison of enzyme activities after adjusting the
amount of purified/crude enzymes so that the final OD value did not
exceed 1.0 at 30 min of incubation.
peroxidation, the IC50 values of MA and OXY were
78.4 mM and 3.6 mM, respectively, and the anti-inflam-
matory effect of OXY was also much greater than that of
MA.19) Moreover, OXY showed approximately 110-fold
higher tyrosinase inhibitory activity than did MA against
L-tyrosine substrate.22) Flavonoids generally exist in
glycosylated form in plant and food sources, and upon
ingestion are absorbed after conversion into deglycosy-
lated aglycones by intestinal enzymes and/or gut
microbial enzymes. Flavonoid aglycones exhibit im-
proved bioavailability and greater bioactivity than the
respective glycosidic forms.23–25) When orally adminis-
tered to rats, MA was metabolized to OXY prior to
absorption into the body,26) and was then transported
into the circulating blood and tissues at high rates.27)
The present study aimed to enhance the tyrosinase
inhibitory activity of MA, found in high concentrations
in MCR water extract, by bioconversion to a more active
oxyresveratrol aglycone using crude enzyme extracts of
lactic acid bacteria. To establish a one-step bioconver-
sion process that yields a product useful without further
purification with chemical compounds or enzymes,
various generally recognized as safe (GRAS) bacteria
were screened to identify those with the highest ꢁ-
glucosidase activity. The MCR extract biotransformed
by crude enzyme extract from Leuconostoc paramesen-
teroides PR (LP) exhibited more potent tyrosinase
inhibitory activity than did untransformed MCR extract
or biotransformed MCR extract prepared using com-
mercially available ꢁ-glucosidase purified from almond.
Biotransformation of Mori Cortex Radicis extract by the crude
enzyme extract of LP. One mL of 20 mg/mL MCR extract in phosphate
buffer (pH 6.0) and 1 mL of crude enzyme extract of LP, as selected in
the ꢁ-glucosidase activity screening, were mixed and the mixture was
incubated at 37 ꢁC for 2 h. Commercial ꢁ-glucosidase from almond
was used as positive control. The activity of the enzyme used was
identical to that of the crude enzyme of LP. After incubation, 1 mL of
the mixture was extracted 3 times with 600 mL of n-butanol (BuOH).
The BuOH layer was dried with
a speed vacuum evaporator
(Modulspin 4080C; BioTron, Bucheon, Korea). The average weight
of the residues was 1.3 mg. The residues were used in an assay for
tyrosinase inhibitory activity and in HPLC analysis.
HPLC and LC/MS analysis. HPLC analysis was performed with a
Dionex P680 HPLC pump and a Dionex UVD170U detector (Dionex
Softron, Germering, Germany). The samples were separated on an
Alltima HP C18 HL column (250 ꢂ 4:6 mm, 5 mm) from Alltech
Associates (Deerfield, IL) and in a Dionex TCC-100 column oven
(Dionex Softron) at 40 ꢁC. The mobile phase consisted of 0.02%
phosphoric acid water (mobile phase A) and MeOH (mobile phase B),
and was run at a flow rate of 1 mL/min on a gradient program, as
follows: 10–20% B (0–10 min), 20–23% B (10–15 min), 23–28% B
(15–25 min), 28% B (25–35 min), and 28–50% B (35–70 min). After
completion of elution, a 15-min reconditioning time was imposed
before the next injection. The samples were dissolved in MeOH, and a
10-mL aliquot of the solution was injected. MA and OXY were
detected at 300 nm.
Materials and Methods
Chemicals and reagents. Mushroom tyrosinase, L-tyrosine, p-
nitrophenyl-ꢁ-D-glucopyranoside (PNPG), kojic acid, phosphoric acid,
and ꢁ-glucosidase from almond were purchased from Sigma-Aldrich
(St. Louis, MO), and OXY (purity ꢀ 95%) was from Sabinsa (East
Windsor, NJ). Methanol (HPLC grade) was from Duksan Pure
Chemicals (Gyeonggi, Korea). The water used in this study was
purified with the Milli-Q system (Millipore, Bedford, MA).
Qualitative analysis for MA and OXY by LC/MS was performed
with an HP 1100 High-Performance Liquid Chromatography (Hewlett-
Packard, Palo Alto, CA) equipped with a QUATTRO LC Triple
Quadrupole Tandem Mass Spectrometer (Micromass, Beverly, MA).
Preparation of mulberroside A-enriched fraction (MEF) by prep-
arative HPLC. The peak at 10.1 min in LC/MS analysis was assumed
to be MA based on the ion peaks at m=z 569 (½M þ Hꢃþ). It was
enriched by preparative HPLC, and the content of the MA in this MA-
enriched fraction (MEF) was verified to be 87.7% (w/w) by analytical
HPLC. The solution of MCR extract was fractionated by BuOH, and
the BuOH layer was evaporated using a speed vacuum evaporator. The
residue was dissolved in MeOH and filtered with a syringe filter
(Millex SLLHR04NL, 0.45 mm PTFE, 4-mm LH; Millipore) to conduct
preparative HPLC. An HPLC pump (model ACME 9000; Younglin
Instrument, Gyeonggi, Korea), a reversed-phase column (21.2 mm ꢂ ꢂ
250 mm, HiQSil C18 HS-10; Kya tech, Tokyo, Japan), UV detector
(UV VIS detector, Younglin Instrument, Gyeonggi, Korea), and
Autochro 3000 software (Gilson, Middleton, WI) were used to isolate
the MA fraction. The mixture of solvent (A) 0.1% TFA water and
solvent (B) MeOH flowed at a rate of 20 mL/min for 70 min with a
linear gradient profile, as described in the HPLC and LC/MS analysis
section above.
Extraction of Mori Cortex Radicis. MCR was purchased from the
Kyung-dong Market in Seoul, Korea. Fifty g of MCR was sliced thinly
with scissors and extracted with distilled water (500 mL) at 50 ꢁC for
3 h. The extract was then filtered through Whatman no. 1 paper to
remove any residue, and the filtrate was dried using a freeze dryer
(TFD5503; Ilshin Bio Base, Gyeonggi, Korea) to obtain dried powder.
Preparation of crude enzyme extracts from lactic acid bacteria. The
bacterial strains used here, which included the species Bifidobacterium,
Lactobacillus, and Leuconostoc, were cultured in MRS medium
containing 0.05% (w/v) L-cysteine HCl at 37 ꢁC anaerobically.
.
Leuconostoc paramesenteroides PR (LP) was isolated from Puerariae
radix as previously reported.28) After 18 h of incubation, the bacterial
cells were collected by centrifugation (2;500 ꢂ g for 20 min at 4 ꢁC)
and the harvested pellet was washed twice with phosphate buffered
saline. The pellets were re-suspended in 100 mM phosphate buffer
(pH 6.0). All of the re-suspended solutions were concentrated at a level
10 times the original cultured volume and then sonicated (VCX 400;
Sonics & Materials, Newtown, CT) with pulsing (2.0 s, 1.0 s) for
30 min in order to extract the enzymes. The disrupted bacterial
solutions were centrifuged at 12;000 ꢂ g for 3 min, and the super-
natants were used as crude enzyme extracts from the microorganisms.
Tyrosinase inhibition assay. Tyrosinase activity using L-tyrosine as
substrate was determined spectrophotometrically by a previously
described method, with slight modifications.29) Nine units of mush-
room tyrosinase in 20 mL of phosphate buffer (20 mM, pH 6.8) was
added to an assay mixture containing 160 mL of 1 mM L-tyrosine
solution and 20 mL of a test sample solution. The L-tyrosine and test
samples were dissolved in 20 mM phosphate buffer (pH 6.8). After
incubation of the reaction mixture at 37 ꢁC for 40 min, the absorbance
of the mixture was measured at 490 nm using a microplate reader. The
ꢁ-Glucosidase activity assay. ꢁ-Glucosidase activity was measured
based on the rate of release of p-nitrophenol from PNPG. Crude
enzyme extracts (90 mL) was pre-warmed to 37 ꢁC for 15 min, and
10 mL of 5.0 mM PNPG was added as substrate. Commercial ꢁ-

Enhanced Tyrosinase Inhibition by Mori Cortex Radicis
1427
Fig. 1. Biotransformational Pathway of Mulberroside A (MA) to Oxyresveratrol (OXY).
inhibitory effects of the samples were represented as % inhibition =
Table 1. ꢁ-Glucosidase Activities of Various Bacterial Strains
(A–B)/A ꢂ 100, where A = net OD490 without a test sample and
B = net OD490 with a test sample. The net OD490 value was the
difference between OD490 after 40 min of reaction and OD490 before
the reaction.
Relative
activity
ꢁ-Glucosidase sources
ꢁ-Glucosidase from almondꢄ
1.000
2.063
1.832
1.770
1.589
0.502
0.470
0.175
0.088
0
Leuconostoc paramesenteroides PR
Bifidobacterium longum subsp. infantis ATCC 15697
B. pseudocatenulatum SJ32
Results and Discussion
B. adolescentis Int57
B. adolescentis ATCC 15703
B. breve ATCC 15700
Screening of bacterial strains suitable for the bio-
transformation of Mori Cortex Radicis
MA, a stilbene glucoside and a major component of
MCR, contains two ꢁ-glucosyl residues, at the C3 and
C40 positions of OXY (Fig. 1). To improve the tyrosi-
nase inhibitory activity of MCR extract by bioconver-
sion of MA to OXY, the ꢁ-glucosidase activities of
various crude enzyme extracts from GRAS microorgan-
isms, especially lactic acid bacteria, were screened by
pNP releasing assay (Table 1). Commercially available
ꢁ-glucosidase from almond was used as positive control.
Among the lactic acid bacteria tested, Leuconostoc
paramesenteroides PR, Bifidobacterium longum subsp.
infantis ATCC 15697, B. pseudocatenulatum SJ32, and
B. adolescentis Int57 showed greater ꢁ-glucosidase
activity than the commercial almond ꢁ-glucosidase
enzyme. Among these, L. paramesenteroides PR (LP),
isolated from Puerariae radix as previously reported,28)
showed the greatest ꢁ-glucosidase activity, 2-fold higher
than that of the positive control. Based on this result, LP
was chosen as a suitable strain for biotransformation of
the MCR extract.
Lactobacillus delbrueckii subsp. delbrueckii ATCC 9649
B. longum RD47
Bifidobacterium sp. RD54
B. longum subsp. longum ATCC 15707
B. bifidum ATCC 29521
B. bifidum BGN4
0
0
0
ꢄPositive control
Relative activities of the various compounds were calculated on the basis of
specific activity adjusted to the concentration of protein, and are represented
as ratios to positive control (1.000).
enzymes, we reasoned that a biotransformation process
based on microbial fermentation without a purification
process of enzyme sources might represent a more
convenient and economical method for the industrial
production of bioactive herbal extracts. Therefore,
suitable microbial strains were investigated to identify
the most efficient organism for the biotransformation of
the MA in MCR extract to OXY. It has been found that
lactic acid bacteria, which are isolated from food sources
and intestinal microbiota, possess ꢁ-glucosidase activity
and can hydrolyze ꢁ-glucosidic bonds in a wide spectrum
of substrates in plants. Biotransformation with an enzyme
extract from lactic acid bacteria would provide not only
a means of converting MA to OXY, but would also yield
a substance safe for use as a skin-whitening agent.
In the present study, the MCR extract biotransformed
by a crude enzyme extract from LP showed a different
HPLC profile from that of the MCR extract biotrans-
formed by commercial almond ꢁ-glucosidase (Fig. 2).
As shown in Fig. 2B, the content of MA in the MCR
extract decreased and the content of OXY increased
after 2 h of biotransformation by LP, but the MCR
extract biotransformed by almond ꢁ-glucosidase showed
Differences in the bioconversion patterns of MA
under treatment by LP crude enzyme extract and
commercial ꢁ-glucosidase
Biotransformation methods using crude enzyme ex-
tracts from GRAS microorganisms have been applied in
various fields, including the food, pharmaceutical, and
cosmetic industries.23,24,30–34) Because the deglucosylated
form of hydroxystilbene has more potent tyrosinase
inhibitory activity than its glucosylated form,22,35) the
biotransformation of stilbene glycoside to an aglycone
has been performed using commercial deglycosylation
enzyme products such as cellulase and Pectinexꢀ.22,36)
Although these products are used widely as hydrolyzing

1428
J. S. KIM et al.
A
B
C
Fig. 2. HPLC Chromatograms of Mori Cortex Radicis (MCR) Extract after Biotransformation.
A, Non-biotransformed MCR extract (control); B, MCR extract biotransformed by crude enzymes from Leuconostoc paramesenteroides PR;
C, MCR extract biotransformed by commercial ꢁ-glucosidase from almond. Elution was monitored with a UV absorption detector at 300 nm.
MA, a peak of mulberroside A (tR, 10.1 min; ESI-MS m=z 569 ½M þ Hꢃþ, 567 ½M ꢅ Hꢃꢅ); OXY, a peak of oxyresveratrol (tR, 25.1 min; ESI-MS
m=z 243 ½M ꢅ Hꢃꢅ); OXY-G, a peak of oxyresveratrol-glucoside (tR, 16.7 min; ESI-MS m=z 405 ½M ꢅ Hꢃꢅ).
A
B
C
Fig. 3. Tyrosinase Inhibitory Effects of Mori Cortex Radicis (MCR) and Related Compounds against Substrate L-Tyrosine.
A, MEF, mulberroside A-enriched fraction; B, Non-biotransformed MCR extract; C, MCR extract biotransformed by crude enzymes from
Leuconostoc paramesenteroides PR (LP). Non-biotransformed MCR extract was exposed to the same environment as the biotransformed MCR
extract except for the use of phosphate buffer in place of the crude enzyme extract from LP. Asterisk means significant difference (p < 0:05)
from negative control in tyrosinase inhibition assay (0% inhibition) by Dunnett’s multiple range test. IC50 values in the tyrosinase inhibition
assay are also represented.
no OXY production after 2 h (Fig. 2C). Instead, its
HPLC profile showed an increased peak at 16.7 min,
which was confirmed by LC/MS analysis (OXY-G,
ESI-MS m=z 405 ½M ꢅ Hꢃꢅ) to be a stilbene glucoside
with one glucosyl moiety, like oxyresveratrol-3-O-ꢁ-D-
glucopyranoside or oxyresveratrol-40-O-ꢁ-D-glucopyra-
noside. The crude enzyme extract from LP hydrolyzed
both glucosidic bonds of MA effectively. Consequently,
most of the MA was biotransformed to OXY within 2 h.
The substrate specificity of the commercial almond ꢁ-
glucosidase was different than that of crude enzyme
extract from LP, and its activity cleaved one glycosidic
bond of MA at a time. After 2 h, 97.1% of the MA from
the MCR extract was biotransformed to OXY by LP,
while only 68.7% of the MA was biotransformed to
OXY-G, and a small amount of OXY was biotrans-
formed by almond ꢁ-glucosidase. Prior to the biotrans-
formation process, no OXY was detected in the MCR
extract (Fig. 2A), but biotransformation by LP increased
the amount of OXY in the MCR extract to 0.36% (w/w),
17.5 times higher than that in the MCR extract
biotransformed by almond ꢁ-glucosidase.
tyrosinase in the range of 0–60 mg/mL.8) In the present
study, the tyrosinase inhibitory activity of the MCR
extract was greater than the value reported by Chang
et al., with an IC50 value of 23.9 mg/mL (Fig. 3B). For
comparative purposes, the tyrosinase inhibitory activity
of kojic acid was assayed as a positive standard. The
IC50 value of kojic acid was found to be 13.1 mg/mL in
our assay system. The content of MA in the MCR
extract was approximately 1.97% (w/w) and no OXY
was detected by quantitative analysis in the present
study. To investigate the tyrosinase inhibitory activity of
MA in MCR extract, a MA-enriched fraction (MEF) of
MCR extract was prepared by preparative HPLC. As
shown in Fig. 3A and B, the IC50 value of MEF was
94.9 mg/mL and the non-biotransformed MCR extract
showed tyrosinase inhibitory activity approximately
4-fold higher than that of MEF. The content of MA in
the MEF and the non-biotransformed MCR extract were
87.7% (w/w) and 1.97% (w/w) respectively. From these
data, it can be inferred that other phytochemicals besides
MA in the extract contributed to the greater levels of
tyrosinase inhibitory activity. Recently, Zheng et al.
compared the tyrosinase inhibitory activity of 29
compounds from Morus nigra roots, and reported that
nine compounds showed greater tyrosinase inhibitory
activity levels than kojic acid.37) Among the compounds
isolated, two chalcones, 2,4,20,40-tetrahydroxychalcone
Enhanced tyrosinase inhibitory activities of MCR
extract biotransformed by LP
Chang et al. have reported that an ethanol extract of
MCR showed a 0–62% inhibitory effect on mushroom

Enhanced Tyrosinase Inhibition by Mori Cortex Radicis
1429
and morachalcone A, showed the highest tyrosinase
inhibitory activity, with IC50 values of 0.062 and
0.14 mM respectively. Because Morus alba and Morus
nigra are of the same genus, it is likely that there are
compounds other than MA and OXY in Morus alba that
exhibit strong tyrosinase inhibitory activity levels in
MCR extract. Hence, we reasoned that the combinatorial
chemistry of crude MCR extract can impart more
benefits by virtue of the combined effects of its various
phytochemicals, although MA itself is a good candidate
for bioconversion into OXY, which possesses highly
potent tyrosinase inhibitory activity. Furthermore, we
found that a simple biotransformation process of whole
MCR extract sufficiently enhanced its tyrosinase inhib-
itory activity. The tyrosinase inhibitory activity of the
MCR extract was increased by approximately 6.5-fold
by biotransformation with a crude enzyme extract from
LP (Fig. 3C). The IC50 value of the transformed MCR
was 3.7 mg/mL, whereas that of the non-transformed
MCR was 23.9 mg/mL.
The enhanced tyrosinase inhibitory activity of the
biotransformed MCR extract due to LP can be attributed
to the increased content of OXY. The strong tyrosinase
inhibitory activity of OXY was previously confirmed in
other research, which found that the IC50 value of OXY
was 0.49–1.2 mM depending on the assay conditions.3,22)
In the present study, the IC50 value of the OXY standard
was 1.1 mM. Among more than 30 stilbene derivatives
that occur naturally in various plants, OXY was reported
to exert higher potent tyrosinase inhibitory activity than
any other stilbene compound. Resveratrol was also
found to exhibit tyrosinase inhibitory activity, but its
IC50 value with a L-tyrosine substrate was 43.5 mM.36)
Resveratrol and its derivatives are abundantly distributed
in plants and dietary food sources, including grapes,
wine, soybeans, and peanuts. While resveratrol glyco-
sides, methoxides, and polymers represent the most
abundant stilbenes in nature, their tyrosinase inhibitory
activities were generally found to be negligible in
comparison to resveratrol and OXY.36,38) Kim et al.36)
reported that piceid, a glucosidic form of resveratrol,
had 8.2-fold less tyrosinase inhibitory activity than
resveratrol. A biotransformed extract of Veratrum
patulum showed an increase of up to 180% in its
tyrosinase inhibitory activity because the concentration
of resveratrol increased after the hydrolysis of piceid
with the use of various commercial enzymes such as
cellulase, ꢁ-glucosidase, dextranase, and amylase. How-
ever, the IC50 value of a non-transformed extract of
Veratrum patulum was 100 mg/mL, and the tyrosinase
inhibitory activity of the biotransformed Veratrum
patulum extract was less than that of a MCR extract
biotransformed by LP.
that study was conducted with purified MA and a
commercial enzyme, we thought that it might be more
helpful to streamline the biotransformation process and
to enhance tyrosinase inhibitory activity without purifi-
cation of OXY. In the present study, we demonstrated
the potential enhancement of tyrosinase inhibitory
activity by a simple biotransformation process using a
crude enzyme extract of LP. After 2 h of biotransforma-
tion, 97.1% of MA from the MCR extract was
biotransformed to OXY by LP, and the content of
OXY in the biotransformed MCR extract increased to
0.36% (w/w). The low IC50 value of the transformed
MCR extract, 3.7 mg/mL, may have resulted from the
increased amount of OXY.
As compared with chemical synthesis and traditional
acid/alkali treatment methods, microbial bioconversion
represents a simple, efficient means of enhancing the
bioactivities of glycones, with benefits including higher
conversion efficiency, fewer by-products, better stereo-
specificity, and the absence of safety concerns. The
present study verified that simple biotransformation
using a crude enzyme extract of lactic acid bacteria was
effective for the production of OXY in a crude extract of
MCR. Furthermore, the whole extract of biotransformed
MCR can serve as a resource for food-grade biomaterial,
offering a sufficiently high level of tyrosinase inhibitory
activity due to the combinatorial chemistry of various
bioactive phytochemicals.
Acknowledgment
This work was supported by the Technology Devel-
opment Program (no. 110124-2-2-SB010) for Agricul-
ture and Forestry of the Ministry for Food, Agriculture,
Forestry, and Fisheries and the Next-Generation Bio-
Green 21 Program (no. PJ008005) of the Rural Devel-
opment Administration of the Republic of Korea.
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