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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).

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.