BioSMART ISSN: 1411-321X

Voume 3, Nomor 1 April 2001

Halaman: 7-13

Inhibitory Activity of Enzymatically Synthesized Polyphenol Glucosides

against Melanogenesis and Mutagenesis

JOKO SULISTYO, ACHMAD DINOTO, RINI HANDAYANI

Balitbang Mikrobiologi, Puslitbang Biologi - LIPI

ABSTRACT

Cyclodextrin glucanotransferase (CGTase) of indigenous microbial strain could synthesis polyphenol glucoside in
the presence of polysaccharides and polyphenol. The inhibitory effect of polyphenol glucoside on melanogenesis that
had been stimulated by the activity of mushroom’s tyrosinase was examined. The polyphenol glucoside inhibited the
mushroom’s tyrosinase higher than those of arbutin did as comparative commercial glucoside did. The inhibitory
effect of polyphenol glucoside on mutagenesis that had been induced by aflatoxin B1 as mutagen was also studied.
The polyphenol glucoside exhibited its capacity as antimutagen was slightly higher than those were arbutin and
aglycone-polyphenol.

Key words: transglucosylation, CGTase, polyphenol glucoside, antimutagenesis, antimelanogenesis.

INTRODUCTION MATERIALS AND METHODS

Polyphenol glucosides with interesting Enzyme preparation

biological activities can be used as biomedicine and
biocosmetic (Funayama, et al. 1994). Chemical
synthesis of such kind glucoside is complicated by
many protection and deprotection steps those are
necessary for regioselective synthesis (Nilson,
1988). Enzymatic synthesis has become more
practical by the use of glycosidases available in
sufficient quantity, since the glycosidases possess
some regioselectivity for hydroxyl linkage to an
acceptor (Murata, et al. 1997).
We have reported enzymatic synthesis of
polyphenol glycosides using cyclodextrin
glucanotransferase (CGTase, EC 2.4.1.19)
extracted from microbial culture filtrate, and its
inhibitory effects on bleaching of β-carotene due to
oxidative activity of linoleic acid (Sulistyo and
Soeka, 2001).
In this study, we examined the
transglucosylation catalyzed by CGTase of Bacillus
polymyxa using soluble starch and polyphenols.
The influence of polyphenol glucosides on
inhibition of the melanogenesis due to tyrosinase
activity and the mutagenesis induced by aflatoxin
B1 were also studied.
The CGTase of Bacillus polymyxa was prepared
by autolysis of intact cells from a culture broth of
B. polymyxa which induced by soluble starch. The
intact cells were washed with distilled water and
the washed cells were left to autolyze at 40oC for
24h. The autolyzate was centrifuged and the
supernatant obtained was dialyzed against 50mM
acetate buffer (pH 6.5). The dialyzed enzyme
solution was incubated at 60oC for 6 h. The heat-
treated soluble enzyme was used as the enzyme
preparation.
Assay of enzyme activity
The CGTase’s activity was assayed by
spectotrophotometric measurement using soluble
starch as a substrate (Funayama, et al. 1993). One
unit of enzyme activity was defined as the amount
of enzyme, which reduced by 0,5 the unit
absorbance at 660 nm per min under the condition.
The reaction mixture (450 µl), containing 0.5%
soluble starch and enzyme in 50 mM acetate buffer
(pH 6.5), was incubated at 40oC for 10 min. The
reaction mixture was added with 1.0 ml of 0.5 N
HCl to stop the enzyme reaction, before addition of
2.5 ml of 0.05% KI solution containing 0.005% I2.
The absorbance at 660 nm was measured after

©2001 Jurusan Biologi FMIPA UNS Surakarta

2 BioSMART Vol. 3, No. 1, April 2001, hal. 7-13

keeping the reaction mixture at room temperature Measurement of inhibitory activity on

for 20 min.
Thin-layer chromatography
Thin layer chromatography (TLC) was carried
out with the ascending method, using silica gel 60
plates (Merck Co., Germany) and n-propanol-water
(85: 15, v/v) as the solvent. Spots were detected by
spraying with 20% H2SO4 in methanol and
subsequent heating at 100 to 150 oC for
approximately 10 min (Sulistyo, et al. 1994; 2000).
Preparation of polyphenol glucoside
CGTase was added to 100 ml of a 50 mM
sodium acetate buffer (pH 6.5) containing 5.0%
soluble starch and 2.5% polyphenols. The reaction
mixture (100 ml) was then concentrated in vacuo to
10 ml. The concentrate was applied to a column of
ODS Wakosil 25 that had been equilibrated with
distilled water containing 0.1% formic acid and
methanol, and the products were eluted with
gradient concentration of methanol to 100%. The
fractions containing polyphenol glucoside being
collected.
tyrosinase
Sodium acetate buffer (0.7 ml of 50 mM, pH
6.5), 1.0 ml of polyphenol glucoside solution and
0.3 ml of tyrosinase were mixed and preincubated
at 25oC for 5 min in a sample cuvette. In a
reference cuvette, the buffer solution was used in
place of tyrosinase. After preincubation, 1.0 ml of
2.5 mM L-DOPA in the buffer was added to each
cuvette and mixed. The increase in absorbance at
480 nm was recorded for 1 min. Using the buffer
without sample of glucoside (Kitao & Sekine,
1994) carried out a control test.
Assay of antimutagenic activity
An aflatoxin B1 solution (0.1 ml, 1 µg/ml) was
mixed with varying concentration of polyphenol
glycoside solution (0.1 ml). A culture broth (10-7)
of mutant Salmonella typhimurium TA98 (0.1 ml)
was added to the reaction mixture and incubated
while shaking at at 37oC for 20 min. Melted soft
agar (2 ml) kept at 45oC was added to the reaction
mixture and immediately poured onto a minimal
glucose agar plate. After 48 h of cultivation on the
plate at 37oC, the number of histidine-independent
(His+) revertants was counted (Ames, et al. 1975;
McCann, et al. 1975).

Figure 1. Thin layer chromatogram of polyphenol glucosides. Transfer products were detected in the presence of
pyrocathecol (Pc), catechin (Ct), hydroquinone (Hq) and resorcinol (Rs). Standard solution (S) containing Maltose
(G-2); Glucose (G-1); Methyl α-glucoside (MG); Arbutin (AR). Pyrocathecol glucoside (PG); catechin glucoside
(CG); hydroquinone glucoside (HG) and resorcinol glucoside (RG). Oligosaccharides (OS-1, OS-2, OS-3).
 SULISTIYO dkk. – Inhibitory Activity of Polyphenol Glucosides Enzymes 3

Table 1. Inhibitory effect of aglycones and glycosides on tyrosinase activity.

Incubation Activity of aglycones on tyrosinase* Activity of glycosides on tyrosinase*

(day) (λ 420 nm) (λ 420 nm)
M-1 M-2 M-3 M-4 M-1 M-2 M-3 M-4
0 0,075 0,079 0,08 0,075 0,019 0,012 0,026 0,018
1 0,133 0,173 0,137 0,128 0,292 0,396 0,479 0,465
2 0,122 0,174 0,116 0,097 0,48 0,511 0,539 0,494
3 0,259 0,298 0,178 0,271 0,574 0,583 0,603 0,52
4 0,341 0,501 0,144 0,346 0,647 0,623 0,533 0,566
5 0,462 0,495 0,143 0,402 0,677 0,602 0,515 0,549

*) Mushrooms ; M-1, Auricularia sp.; M-2, Pleurotus sp.; M-3, Lentinus sp.; M-4, Ganoderma sp.

RESULTS AND DISCUSSION The inhibitory effect of polyphenol on

The polyphenol glucosides were synthesized by
incubating soluble starch and each acceptors such
as hydroquinone, pyrocathecol, resorcinol and
catechin, with CGTase at 40ΒC for 24 h. To
convert oligoglucoside, amyloglucosidase was then
added to the reaction mixture (Figure 1). Rf value
of the transfer products those were pyrocathecol
glucoside (0.75), catechin glucoside (0.77),
hydroquinone glucoside (0.73) and resorcinol
glucoside (0.75) were comparable to the Rf value
of arbutin-standard (0.81), while pyrocathecol
glucobioside (0.68) and resorcinol glucobioside
(0.67) were lower than those of Rf value of
glycoside standards those were 0.81 and 0.63,
respectively.
melanogenesis in-vitro was investigated
spectrophotometrically, by monitoring the
increasing absorbance caused by conversion of
tyrosine to dopachrome by autooxidation.
Tyrosinase catalyzes the formation of melanin from
tyrosine via 3,4-dihydroxy-phenylalanine (DOPA)
and dopaquinone. In the course of screening of an
effective tyrosine inhibitor, we found that the
aglycone-polyphenol (A=0.127, at 420 nm)
inhibited the activity tyrosinase from mushrooms
more effective until three days of incubation than
those of polyphenol glycosides (A=0.506, at 420
nm). The inhibitory activity of purified polyphenol
glycoside against tyrosinase activity was
determined as shown in Table. 1.

Tiro sinase

OH OH O

O2 O2 M elanin
OH O
HO O C NH 2 HO O C NH 2 HO O C NH 2
Tyros ine DO PA Dopa quinone Tyros ina se

OH O
OH OH
O OH
Red dis h
O2
O2
OH O
O OH THB HBQ
BQ HQ

BQ HQ

Figure 2. Browning resistance and tyrosinase inhibition activity of aglycones and polyphenol glycosides.
4 BioSMART Vol. 3, No. 1, April 2001, hal. 7-13

1,8 120
Absorbance at 460 nm
1,6
100

Browning Resistance
1,4 % Inhibitory

1,2 80
1
60
0,8
0,6 40
0,4
20
0,2
0 0
Hq Pc Rs Ar HqG PcG RsG
Polyphenolic Compounds

Figure 3. Inhibition mechanism of polyphenol on melanogenesis.

Browning resistance and inhibitory effects of The inhibitory effect of polyphenol on

some polyphenol glucosides against activity of melanogenesis in-vitro was investigated
tyrosinase were investigated and compared with spectrophotometrically, by monitoring the
those arbutin and aglycones (hydroquinone, Hq; increasing absorbance caused by conversion of
pyrocathecol, Pc; and resorcinol, Rs). The tyrosine to dopachrome by autooxidation.
inhibitory effects of polyphenol glycosides Tyrosinase catalyzes the formation of melanin from
(hydroquinone-glucoside, HqG; pyrocathecol- tyrosine via 3,4-dihydroxy-phenylalanine (DOPA)
glucoside, PcG; resorcinol-glucoside, RsG,) and and dopaquinone. In the course of screening of an
arbutin (Ar) on tyrosinase activity were effective tyrosine inhibitor, we found that the
comparable. Although the inhibitory activities of aglycone-polyphenol (A=0.127, at 420 nm)
these glycosides were slightly lower than those inhibited the activity tyrosinase from mushrooms
were aglycones, however, the glycosides were more effective until three days of incubation than
more stable against browning than those of those of polyphenol glycosides (A=0.506, at 420
aglycones (Figure 2). nm). The inhibitory activity of purified polyphenol
glycoside against tyrosinase activity was
determined as shown in Table. 1.

350
310 310 310
300
Mutation
Colony Counting

250 Control
25 mM
200 50 mM
90 mM
150

100
66 62 57
56 56 56
50 36
1 0 6 5
0
Arbutin Polyphenol Glucoside Aglycone
Polyphenolic Compounds

Figure 4. Inhibitory effect of polyphenolic compounds towards mutagenesis induced with aflatoksin-B1.
 SULISTIYO dkk. – Inhibitory Activity of Polyphenol Glucosides Enzymes
120
100 100
100
Mutagenesis (%)
80
60
47.63
40
20
0
0 1
Antimutagen Concentration (mM)
Figure 5. Effect of polyphenol glucoside compared to arbutin towards mutagenecity of aflatoksin-B1 with S.
typhimurium.
Browning resistance and inhibitory effects of
some polyphenol glucosides against activity of
tyrosinase were investigated and compared with
those arbutin and aglycones (hydroquinone, Hq;
pyrocathecol, Pc; and resorcinol, Rs). The
inhibitory effects of polyphenol glycosides
(hydroquinone-glucoside, HqG; pyrocathecol-
glucoside, PcG; resorcinol-glucoside, RsG,) and
arbutin (Ar) on tyrosinase activity were
comparable. Although the inhibitory activities of
these glycosides were slightly lower than those
were aglycones, however, the glycosides were
more stable against browning than those of
aglycones (Figure 2).
In the presence of intermediator (DOPA), the
tyrosinase is activated towards polyphenol as
poorly effective substrate. That the fast oxidation
of polyphenol (HQ) in the presence of catalytic
DOPA is the result of a true enzymatic reaction.
This would ensure that dopaquinone formed
enzymatically is reduced back to DOPA by HQ, so
that the constant level of cofactor is available to
keep tyrosinase activated (Figure 3).
Many epidemiological studies have been carried
out to find causal relationship between cancer
prevention and consumption of tea which
containing polyphenol. It was reported that mice
which were fed with polyphenol containing diet,
the growth of implanted tumor cells was
tremendously compressed. Tropical application of
green tea polyphenol fraction inhibited 12-O-
tetradecanoylphorbol-13-acetate-induced tumor
promotion in mouse skin.
In the present study, we examined the
suppressive activity of polyphenol glycoside
5
Arbutin
Polyphenol glucoside
52.68
46.69
43.53
33.4433.75
23.9725.87
5 10 15
toward the mutagenicity of aflatoxin B1 with
Salmonella typhimurium TA98 in the absence of
S9 mix. S. typhimurium TA98 is a mutant of
Salmonella bacteria that requires histidine to grow
(his-), owing to mutation in a gene for histidine
biosynthesis. The activated mutagen induces
reverse mutation, and resulting wild-type revertants
(his+) can grow in the histidine-deficient medium.
By this method, we observed that aglycone,
glycosides and arbutin have a potent inhibitory
effect on the mutagenicity of aflatoxin B1 (Figure
4).
Heterocyclic amines and polyaromatic
hydrocarbons are considered to be the major cause
of cancer due to their potent carcinogenicity. They
are formed during the daily cooking and commonly
occur in food. Burnt grilled fish or smoked foods
contain a mutagenic heterocyclic amine, 3-amino-
1,4-dimethyl-5H-pyridol[4,3-b]indole (Trp-P-1).
The Trp-P-1 acquires genotoxicity after being
converted to N-hydroxy Trp-P-1 by hepatic
enzyme cytochrome P450 monooxygenases
(P450). N-hydroxy Trp-P-1 is easily transformed to
its radical, which reacts with DNA to induce frame-
shift mutation.
In another present study, we examined the
suppressive activity of polyphenol glucoside
compared to arbutin towards the mutagenicity of
aflatoxin B1 with Salmonella typhimurium TA98 in
the absence of S9 mix. Eventhough, the
antimutagenic activity of polyphenol glycoside was
found to be slight different to that of aglycone, but
it was found comparably to that of arbutin. This
result suggested that polyphenol glycoside
possessed considerable antimutagenic effects on
6 BioSMART Vol. 3, No. 1, April 2001, hal. 7-13
the transition-type mutation than on the frame shift
type. Furthermore, it is suggested that polyphenol
glycoside inhibited the metabolic enzyme
converting aflatoxin B1 to mutagenic forms.
CONCLUSION
Polyphenol is one of the most effective
inhibitors of melanogenesis in vitro, and is widely
used for the treatment of melanogenesis and other
hyperpigmentory disorders. However, polyphenols
are limited used since they are easily degraded in
an aqueous solution resulting in rapid. In attempt to
synthesis more stable glucoside linked polyphenol,
we have synthesised the polyphenol glucosides by
application of transglucosylation of CGTase from
indigenous microbial culture filtrate. The transfer
products were furthermore examined to study the
inhibitory activity against melanogenesis due to
tyrosinase activity. Suppressive activity of
polyphenol glucoside towards the mutagenicity
induced by aflatoxin B1 on Salmonella typhimurium
TA98 was also studied. We have found that the
polyphenol glucoside exhibited its biological
properties, that may be advantageous for
biopharmaceutical and cosmetical application
purposes as bioantimutagen and bioantimelanogen.
ACKNOWLEDGEMENT
We would like to acknowledge and appreciate
the Committee of RUT project, Ministry of Science
and Technology, LIPI and BPPT for valuable
research grant.
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