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The
Synthesis of two Arbutin Derivatives and Inhibitory Effect of Them
on Mushroom Tyrosinase.
Abstract: Tyrosinase (1.14.18.1) is a metalloenzyme oxidase, known
as a key enzyme in melanin biosynthesis, involved in determining the
color of mammalian skin and hair. Its abnormal expressing would
contribute to various dermatological disorders, such as melasama,
age spots, and sites of actinic damage. The inhibitors of tyrosinase
can become potent whitening agents to meet the medical requirements
for depigmenting agents. Arbutin is a cosmetic additive for
whitening, according to that it can inhibit tyrosinase activity. In
this paper, two kinds of arbutin derivatives were synthesized
derived from D-glucose. The deritives were p-isopropylphenyl D-glycopyranose
and p-methoxyphenyl D-glycopyranose. Adding to triethylamine and BF3·ether,
1’,2’,3’,4’6’-penta-O-acetyl-D-glycose
was reacted with p- isopropylphenol and p-methoxyphenol to afford
the target product, respectively. The structure of products were
identified by IR spectra. Thesis three compounds acted as effector
study the inhibit effect of them on mushroom tyrosinase when L-DOPA
is substrate. It can help study deeply the relationship between the
structure of inhibitors and the inhibitor capacity of them on the
tyrosinase. The result elucidated the sequence of inhibitor
capacity: arbutin＞p-isopropylphenyl
D-glycopyranose＞p-methoxyphenyl
D-glycopyranose. It showed that the hydroxy group on the benzene
ring acted important effect on inhibitory capacity, which according
with the result reported that arbutin was a competitive inhibitor.
Arbutin (hydroquinone-D-glucopyranoside)
is an abundant solute in the leaves of many freezing- or
desiccation-tolerant plants. Its physiological role in
plants, however, is not known. Here we show that arbutin
protects isolated spinach (Spinacia oleracea L.)
thylakoid membranes from freeze-thaw damage.
During freezing
of liposomes, the presence of only 20 mM arbutin led to
complete leakage of a soluble marker from egg PC (EPC)
liposomes. When the nonbilayer-forming chloroplast lipid
monogalactosyldiacylglycerol (MGDG) was included in the
membranes, this leakage was prevented. Inclusion of more
than 15% MGDG into the membranes led to a strong
destabilization of liposomes during freezing. Under these conditions
arbutin became a cryoprotectant, as only 5 mM arbutin reduced
leakage from 75% to 20%.
The nonbilayer lipid
egg phosphatidylethanolamine (EPE) had an effect similar
to that of MGDG, but was much less effective, even at
concentrations up to 80% in EPC membranes. Arbutin-induced
leakage during freezing was accompanied by massive
bilayer fusion in EPC and EPC/EPE membranes. Twenty percent MGDG
in EPC bilayers completely inhibited the fusogenic effect of
arbutin.
The membrane surface
probes merocyanine 540 and
2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine
(NBD-C6-HPC) revealed that arbutin reduced the
ability of both probes to partition into the membranes.
Steady-state anisotropy measurements with probes that
localize at different positions in the membranes showed
that headgroup mobility was increased in the presence of
arbutin, whereas the mobility of the fatty acyl chains
close to the glycerol backbone was reduced. This reduction,
however, was not seen in membranes containing 20% MGDG.
The effect
of arbutin on lipid order was limited to the interfacial region
of the membranes and was not evident in the hydrophobic core
region. From these data we were able to derive a physical
model of the perturbing or nonperturbing interactions of
arbutin with lipid bilayers.
Arbutin
(4-hydroxyphenyl-D-glucopyranoside)
is a glycosylated hydroquinone (Fig.
1) that has been found at
extraordinarily high concentrations in the leaves of
several plant species, such as Vaccinium spp. (Suau
et al., 1991). It
has been used pharmaceutically in humans for centuries,
either as plant extracts or, in more recent decades, in
purified form, because of its diuretic and urinary
antiinfective properties. There is nothing known about
the physiological role of arbutin in the plants that synthesize
it. The tolerance of many of these plants against
environmental stresses such as frost and drought,
however, could be related to the presence of arbutin.
This is especially striking in the
resurrection plant Myrothamnus flabellifolia, where arbutin
constitutes as much as 25% of the dry weight of the
leaves,which, assuming a uniform distribution in the
cells, translates into a concentration of ~100 mM. This
concentration would of course be higher if arbutin were restricted
to specific cellular compartments. Resurrection plants are
able to survive complete dehydration for extended periods
of time. Although the physiological mechanisms underlying
desiccation tolerance have not been completely
understood, the accumulation of soluble sugars and other
solutes, such as arbutin, is widely recognized as an
important part of the cellular stress protection in plants.
This is true for desiccation and freezing, as
plants employ similar biochemical adaptations to cope
with the two stresses. This can be rationalized from the
fact that during freezing, ice crystallization leads to an
effective removal of liquid water and consequently to
freeze-induced dehydration. Therefore, over a wide range
of temperatures/water contents, freezing and desiccation
challenge cellular structures with the same physical
stresses. Only at the extremes of desiccation, when more
cellular water is removed than would crystallize during
freezing under physiologically relevant conditions, would the
two treatments result in physically different stresses
FIGURE1
Line drawing of the chemical structure of arbutin (4-hydroxyphenyl-D-glucopyranoside).
A possible function of arbutin in plant stress
tolerance could be the inhibition of membrane degradation in partly
or completely desiccated or frozen leaves. Ioku et al.
(1992)showed that arbutin has antioxidative properties for
membrane lipids, and Oliver et al. (1996)reported that it can inhibit the enzyme phospholipase A2
(PLA2) in partially dehydrated liposomes. This inhibitory
activity is most likely mediated by a direct interaction
of arbutin with the lipid bilayer (Oliver et al., 1998),
which is already seen with completely hydrated membranes
and is probably enhanced when water is removed. In both
hydrated and dry bilayers, made from different, pure
species of phosphatidylcholine, the phase transition
temperature between the gel and liquid-crystalline phases (Tm)
is significantly reduced in the presence of arbutin,indicating
that its interaction with membranes has an influence on
the physical properties of the membrane lipids.
Surprisingly, this interaction leads to a
destabilization of large unilamellar PC vesicles during drying
(Oliver et al., 1998).
This finding is obviously at odds with the proposed role of
arbutin in plant stress tolerance. It should be
recognized, however, that pure phospholipid vesicles
might not be an ideal model system for the study of the
stress tolerance of plant membranes. Because the
intracellular localization of arbutin has not been determined
in any plant and consequently its natural target membranes are
unknown, we decided to use isolated chloroplast thylakoid
membranes from spinach as a well-defined experimental
target membrane for arbutin. Because freeze-thaw damage
to thylakoids (Hincha et al., 1996)
and to liposomes containing different thylakoid lipids (Hincha
et al., 1998)
has been extensively studied, we have used the resulting
knowledge to investigate the effects of arbutin on the freeze-thaw
stability of membranes.
Differences from results obtained with
pure PC membranes could be expected, because thylakoids contain
mostly glycolipids. The nonbilayer lipid
monogalactosyldiacylglycerol (MGDG) accounts for ~50% of
the thylakoid lipid content, while the bilayer lipids
digalactosyldiacylglycerol (DGDG) (~25%), phosphatidyl-glycerol
(PG) (~15%), and sulfoquinovosyldiacylglycerol (SQDG) (~10%)
make up the other half (Webb and Green, 1991).
In the present paper we show that arbutin is a cryoprotectant for
thylakoid membranes and that the cryoprotective effect is
dependent on the presence of MGDG in large unilamellar liposomes.
Results from experiments with several membrane probes indicate
that arbutin influences the physical state of the membrane
lipids in the headgroup and interfacial regions and that
there are characteristic differences between membranes
that contain MGDG and those that do not.
MATERIALS AND METHODS:
Lipids and membrane probes
Phospholipids were purchased from Avanti Polar Lipids (Alabaster,
AL) or from Sigma. Galactolipids were purified as described
in detail in recent publications (Hincha and Crowe, 1996;
Hincha et al., 1998),
from fresh spinach (Spinacia oleracea L.) leaves
obtained from a local market in Davis, CA. Alternatively, MGDG
and DGDG from soybean leaves were purchased from Lipid
Products (Redhill, UK).
There were no detectable differences in our experiments
in the effects of arbutin on liposomes made with the lipids from
different sources. Carboxyfluorescein (CF) was obtained from
Molecular Probes (Eugene, OR) and was purified according
to the procedure described by Weinstein et al. (1984).N-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine
(NBD-PE), N-(lissamine rhodamine B
sulfonyl)-dioleoylphosphatidylethanolamine (Rh-PE),
2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine
(NBD-C6-HPC), and
trimethylammoniumpropyl-1,6-diphenyl-1,3,5-hexatriene (TMAP-DPH)
were purchased from Molecular Probes. Merocyanine 540 (MC540),
1,6-diphenyl-1,3,5-hexatriene (DPH), and trimethylammonium-DPH
(TMA-DPH) were obtained from Sigma.
Preparation of liposomes:
The different lipids
were mixed in chloroform, dried under a stream of N2, and
stored under vacuum overnight to remove traces of
solvent. Mixtures of different lipids were made by weight and
are expressed as a percentage (w/w). All liposomes were
prepared from hydrated lipids, using a hand-held extruder
with two layers of polycarbonate membranes (Poretics,
Livermore, CA) with 100-nm pores.
Liposome freezing experiments:
Liposomes (20 µl) were mixed with an equal volume
of concentrated solutions of arbutin made in 10 mM TES, 0.1 mM EDTA,
50 mM NaCl (TEN buffer, pH 7.4) (final lipid
concentration 5 mg ml1).
The tubes were placed in a bath containing ethylene glycol
cooled to 18°C. After
5 min the samples were crystallized by touching the
outside of the tubes with a spatula cooled in liquid
nitrogen. The samples were kept frozen for 3 h and thawed in a
water bath at room temperature. Control samples were incubated
at 0°C for 3 h. Freeze-thaw damage was determined either as
leakage of the soluble marker CF or as membrane fusion.
The figures show the means ± SD from three parallel
samples. Where no error bars are visible, they were
smaller than the symbols.
Determination of freeze-thaw damage to thylakoids:
Thylakoids were isolated from spinach leaves
as described previously. The membranes were washed three times in
10 mM MgCl2, 20 mM K2SO4.
Samples (0.2 ml) containing ~0.5 mg chlorophyll ml1, 5 mM
MgCl2, 10 mM K2SO4, 150 mM
K-glutamate, 50 mM sucrose (artificial stroma medium),
and additional arbutin were placed in a freezer at20°C for 3 h
and were rapidly (within 2-3 min) thawed in a water bath
at room temperature. Control samples were kept for the
same time at 0°C. After thawing, the membranes were
sedimented by centrifugation (15 min at 16,000 × g), and
the supernatants were mixed with an equal volume of
electrophoresis sample buffer (Laemmli, 1970).
Proteins were fractionated on 15% acrylamide sodium
dodecyl sulfate-polyacrylamide gel electrophoresis gels
under reducing conditions and were then transferred to
nitrocellulose membranes by electroblotting (Towbin et
al., 1979).
Unoccupied binding sites on the membranes were blocked by
incubation in 5% (w/v) milk powder, 0.1% (v/v) Tween
20 in 25 mM Tris, and 150 mM NaCl (pH 7.5; Johnson et
al., 1984).
Filters were probed with rabbit anti-spinach plastocyanin
antiserum. Bound IgG on the filters was visualized with a
goat anti-rabbit IgG serum labeled with horseradish
peroxidase (BioRad) as described by Sieg et al. The
stained bands were quantified with a laser densitometer.
For comparison with the frozen-thawed samples,
plastocyanin was completely liberated from thylakoids by sonication
with a tip sonicator for 5 min at 80 W. The membranes were
removed by centrifugation (30 min at 20,000 × g),
and the supernatant was treated as described
above.
Partitioning of merocyanine 540 and NBD-C6-HPC into
liposome membranes:
To assess the effects of arbutin on the
surface properties of membranes, we used the dye MC540 as described
by Bakaltcheva et al. (1994)and the fluorescent probe NBD-C6-HPC as described
by Lee and Lentz (1997).
For MC540 measurements, liposomes (0.3 mg ml1)
were suspended in TEN containing up to 200 mM arbutin. Samples
were incubated at 0°C for 30 min, and then MC540 was added to
a final concentration of 105
M. After 15 min, the absorbance was measured at 570 nm and 530 nm
on a Uvikon 922 double-beam spectrophotometer (Kontron Instruments,
Neufahrn, Germany) at room temperature. The reference cuvette
contained liposomes and arbutin without MC540. The data were
corrected for the effect of arbutin on the absorbance of
MC540 in the absence of liposomes.
The increase in fluorescence emission from NBD-C6-HPC
resulting from the partitioning of the fluorescently labeled lipid
into preformed liposomal membranes was measured with a
Kontron SFM 25 fluorometer at 25°C. Liposomes (0.1 mg ml1)
were suspended in a cuvette in TEN containing different
concentrations of arbutin. NBD-C6-HPC was
added as a concentrated solution in methanol to a final
lipid/probe ratio of 200:1 and a final methanol
concentration of 0.1% (v/v). The resulting fluorescence emission
was measured at 530 nm, with excitation at 470 nm. The data at
all arbutin concentrations were corrected for the fluorescence
of NBD-C6-HPC in the absence of liposomes (under
1%). Arbutin had no measurable influence on the
fluorescence emission of the probe in the absence of
membranes.
Steady-state anisotropy of membrane lipids:
The dynamics of lipids in liposome membranes in the presence of
different concentrations of arbutin was determined by measuring
the degree of depolarization of the fluorescence emitted from
the probes DPH, TMAP-DPH, TMA-DPH, and NBD-PE (Lentz, 1993).
DPH is a hydrophobic molecule and is widely used for
measuring the order of the lipid fatty acyl chains in the
core region of the bilayer, whereas TMAP-DPH and TMA-DPH
are anchored at the water/lipid interface, because of
their additional charged trimethylammonium group (Engel
and Prendergast, 1981;
Prendergast et al., 1981).
NBD-PE is an indicator of the mobility of the lipid headgroup
region of the membranes (Lentz et al., 1996).
DPH, TMAP-DPH, or TMA-DPH in dimethyl formamide was added
to a liposome suspension (0.1 mg ml1) in
TEN containing up to 200 mM arbutin in a stirred cuvette
at 25°C. The lipid/probe ratio was 200:1, and the final dimethyl
formamide concentration was 0.1% (v/v). Measurements were
carried out on a Kontron SFM 25 spectrofluorimeter with
polarization filters. Fluorescence was excited at 360 nm,
and emission was recorded at 450 nm. NBD-PE in chloroform
was mixed with the other lipids at a lipid/probe ratio of
200:1, and liposomes were prepared in TEN as described
above. The liposomes were suspended in TEN and arbutin as
above, and fluorescence depolarization was measured at an
excitation wavelength of 470 nm and an emission wavelength
of 530 nm.
RESULTS:
To investigate the
effects of arbutin on the stress tolerance of plant membranes, we
have frozen isolated spinach thylakoids in a simplified
artificial stroma medium, which elicits freeze-thaw
damage similar to that in the in vivo situation. As a molecular
marker for membrane damage we used plastocyanin, a
soluble electron transport protein that is localized in the
lumen of thylakoid vesicles. Its appearance in the supernatant
of membrane samples after centrifugation is closely related to
the inactivation of photosynthetic electron transport, in both
leaves and isolated thylakoids, and indicates transient
membrane rupture.
As a first step in the analysis, the nonbilayer lipid MGDG was
chromatographically separated from the bilayer lipids DGDG,
PG, and SQDG, constituting the DG+ fraction (Hincha et al.,
1998).
The lipids were then reconstituted into liposomes containing
80% DG+ and either 20% egg phosphatidylcholine (EPC) or
MGDG. A freeze-thaw cycle induced CF leakage that
depended on both the lipid composition and the
concentration of arbutin (Fig. 2). As described in detail
before (Hincha et al., 1998),
MGDG destabilizes membranes and leads to increased
leakage during freezing. Concentrations higher than 20%
MGDG in the membranes result in increased leakage, even
in the absence of an additional stress.
FIGURE 2
Freeze-thaw damage to large unilamellar liposomes in the presence of
arbutin.Liposomes were prepared from mixtures of 80% MGDG-depleted
chloroplast lipids (DG+) and 20% EPC or 20% MGDG.The samples were
frozen at 18°C for
3 h. Freeze-thaw damage was determined as leakage of the soluble
marker carboxyfluorescein (CF).
FIGURE 3 CF leakage from liposomes in the
presence of different concentrations of arbutin during freezing. The
membranes were composed of EPC and different fractions of MGDG.
FIGURE 4
Freeze-thaw damage to large unilamellar liposomes in the presence of
arbutin.Liposomes were prepared from mixtures of 80% MGDG-depleted
chloroplast lipids (DG+) and 20% EPC or 20%MGDG.The samples were
frozen at18°C for
3 h. Freeze-thaw damage was determined as leakage of the soluble
marker carboxyfluorescein (CF).
FIGURE 5
Freeze-thaw damage to liposomes in the presence of arbutin. The
membranes were composed of either 50% EPC and 50% DGDG, or 30% EPC,
50% DGDG, and 20% MGDG.
FIGURE 6
CF leakage from liposomes in the presence of different
concentrations of arbutin during freezing. The membranes were
composed of EPC and different fractions of
EPE.
FIGURE 7
Freeze-thaw damage to liposomes in the presence of different
concentrations of arbutin during freezing, measured as bilayer
fusion. The membranes were composed of EPC and different fractions
of either MGDG or EPE.
FIGURE 8
Absorbance ratio A570/A530 of
MC540 in the presence of liposomes of different composition and
different concentrations of arbutin. A reduction in the absorbance
ratio indicates reduced partitioning of the dye into the lipid
headgroup region of the membranes. The means ± SD of three parallel
samples are shown.
FIGURE 9
Fluorescence emission of the lipid probe NBD-C6-HPC in
the presence of liposomes of different composition as a function of
the concentration of arbutin. Reduced fluorescence emission
indicates a reduced ability of the probe to partition into the
membranes. The means ± SD of three parallel samples are shown.
FIGURE 10
Steady-state fluorescence anisotropy of NBD-PE in liposomes of
different composition as a function of the concentration of arbutin.
NBD-PE reports on the fluidity in the lipid headgroup region of the
membranes. The means ± SD of three parallel samples are shown.
FIGURE 11
Steady-state fluorescence anisotropy of TMA-DPH, TMAP-DPH, and DPH
in liposomes of different composition as a function of the
concentration of arbutin. The symbols are the same as in Fig. 11 and
indicate the means ± SD of three to six parallel samples. The probes
report on the dynamics of the lipids in the hydrophobic core region
of the membranes (DPH) or progressively closer to the
membrane-solution interface (TMAP-DPH, TMA-DPH). For TMAP-DPH the
differences for each lipid composition between samples in the
absence of arbutin and samples containing 200 mM arbutin were
significantly different at p = 0.0005 in a t-test. For
TMA-DPH significant differences were found for EPE (p = 0.0005)
and EPC (p = 0.01). No significant difference was found for
liposomes containing MGDG (p > 0.375).
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FIGURE 12
Schematic representation of cross sections of the outer monolayer of
liposome membranes containing either only EPC (cylindrically
shaped molecules) or a mixture of EPC and MGDG (cone-shaped
molecules) in the absence and presence of arbutin. Darkly shaded
parts of the lipids represent the headgroups, lightly shaded parts
the acyl chain regions. Arbutin is depicted as two elipses, with the
lower part representing the phenol ring and the upper part the
glucose moiety (compare Fig. 1).
The Synthesis of two Arbutin Derivatives and Inhibitory Effect of
Them on Mushroom Tyrosinase:
Arbutin is a cosmetic additive for whitening,
according to that it can inhibit tyrosinase activity. In this paper,
two kinds of arbutin derivatives were synthesized derived from
D-glucose. The deritives were p-isopropylphenyl D-glycopyranose and
p-methoxyphenyl D-glycopyranose. Adding to triethylamine and BF3·ether,
1’,2’,3’,4’6’-penta-O-acetyl-D-glycose
was reacted with p- isopropylphenol and p-methoxyphenol to afford
the target product, respectively. The structure of products were
identified by IR spectra. Thesis three compounds acted as effector
study the inhibit effect of them on mushroom tyrosinase when L-DOPA
is substrate. It can help study deeply the relationship between the
structure of inhibitors and the inhibitor capacity of them on the
tyrosinase. The result elucidated the sequence of inhibitor
capacity: arbutin＞p-isopropylphenyl
D-glycopyranose＞p-methoxyphenyl
D-glycopyranose. It showed that the hydroxy group on the benzene
ring acted important effect on inhibitory capacity, which according
with the result reported that arbutin was a competitive inhibitor.
Arbutin
(Beta Arbutin)
Chemical Name:
4-hydroquinone--D-glucopyranoside
Molecular formula:
C12H16O7
Molecular weight:
272.25
Structure:
Arbutin (beta-arbutin)is
a natural double action whitening agent.
Arbutin (Beta Arbutin)
is a new type of skin de-pigmentation and whitening agents, an
extract of Bearberry plant which produced by a solid /liquid
extraction, an environmentally friendly process .
Recommendation on use of arbutin:
1. Arbuin is prone to
hydrolysis under acidic condition. It shall be used at pH 7.0-7.1.
2. Antioxidant and
decolorant are required in cosmetic preparations.
3. Add 0.8-1.0% of
azone to enhance dermal absorption.
The Arbutin is used for be making up
the industry:
Arbutin was first
discovered in Arctostapylos uva-ursi Spreng and then in the leaves
of Vaccinicum vitis-idaca L., Pyrus pyrifolia Kakai. and Saxifraga
stolonifera (L.) Meerb. It is used as additive of drug and
cosmetics. It can relieve cough, remove the phlegm, diminish
inflammation etc.. So it is used as antitussive, urethra
disinfector, also as food extender in the US.
It may be used to
repress the virulence of bacterial pathogens and to prevent
contaminating bacteria, it is also used for treating allergic
inflammation of the skin . More recently, Arbutin has been used to
prevent pigmentation and to whiten the skin beautifully. It can be
used to whiten the skin, to prevent liver spots and freckles, to
treat sunburn marks and to regulate melanogenesis.
Arbutin is very safe
skin agent for external use which does not have toxicity ,
stimulation, unpleasant odor or side effect such as Hydroqinone.The
encapsulati on of Arbutin constitute a delivery system to
potentialize the effect in time. It is a way to incorporate the
hydrophilic Arbutin in lipophilic media. Arbutin give three main
properties; Whitening effects, anti- age effect and UVB/ UVC filter.
[Arctostaphylos uva-ursi (Bearberry)]:
This plant from the
botanical family of the ericacae grows in many areas of the world,
in Europe, in Northern Temperate Asia and north America.
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熊果素.
