Article

Inhibitory Effects of .ALPHA.-Arbutin on Melanin Synthesis in Cultured Human Melanoma Cells and a Three-Dimensional Human Skin Model

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Abstract

We studied the inhibitory effects of 4-hydroxyphenyl alpha-glucopyranoside (alpha-arbutin) on melanogenesis in cultured human melanoma cells, HMV-II, and in a three-dimensional cultured human skin model. alpha-Arbutin showed no inhibitory effect on HMV-II cell growth at a concentration below 1.0 mM. Melanin synthesis in cells treated with alpha-arbutin at 0.5 mM decreased to 76% of that in non-treated cells. The cellular tyrosinase activity of HMV-II cells also significantly decreased, while the expression of its mRNA was not affected. Melanin synthesis in a human skin model was also evaluated by the macro- and microscopic observation of its pigmentation as well as by quantitative measurements of melanin. Treatment of the human skin model with 250 microg of alpha-arbutin did not inhibit cell viability, while melanin synthesis was reduced to 40% of that in the control. These results indicate that alpha-arbutin is an effective and safe ingredient for skin-lightening.

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... Arbutin (hydroquinone-O-β-D-glucopyranoside), a natural polyphenol isolated from the bearberry plant Arctostaphylos uvaursi, has been traditionally used as a whitening agent (8,9). Arbutin has been found to reduce the melanin content and tyrosinase activity in cultured human melanocytes, B16 murine melanoma cells and HMV-II cells (10)(11)(12)(13)(14). It has been reported that certain arbutin derivatives exert more prominent inhibitory effects on melanin content and tyrosinase activity compared with those of arbutin (12,15,16). ...
... Arbutin has been found to reduce the melanin content and tyrosinase activity in cultured human melanocytes, B16 murine melanoma cells and HMV-II cells (10)(11)(12)(13)(14). It has been reported that certain arbutin derivatives exert more prominent inhibitory effects on melanin content and tyrosinase activity compared with those of arbutin (12,15,16). Furthermore, arbutin has been confirmed to exert a pro-apoptotic effect on human bladder cancer TCCSUP and human melanoma A375 cells (17,18). ...
... Following incubation with arbutin 6'-undecenoate, melanin production decreased by ~70% in B16 melanoma cells compared with that in control cells (31). In the present study, the acetylated arbutin derivative exhibited a higher efficacy in terms of tyrosinase activity inhibition and reduction of melanin production compared with arbutin, which may be associated with the increased solubility in oil-based systems and the improvement of membrane penetration (10)(11)(12). ...
Article
Arbutin, a natural polyphenol isolated from the bearberry plant Arctostaphylos uvaursi, possesses whitening and anticancer properties. The effects of arbutin on melanogenesis and its pro-apoptotic effect on B16 murine melanoma cells have not yet been reported. In the present study, acetylated arbutin was prepared in order to improve the biological effects of arbutin, and it was found to significantly inhibit the biosynthesis of melanin and tyrosinase activity compared with parent arbutin in B16 murine melanoma cells. Interestingly, only acetylated arbutin strongly inhibited B16 murine melanoma cell migration in a dose-dependent manner. Both arbutin and acetylated arbutin significantly reduced cell viability, promoted cell apoptosis, caused G1 cell cycle arrest and induced mitochondrial disruption in B16 murine melanoma cells. Furthermore, reduced expression of B-cell lymphoma‑extra large (Bcl-xL) and Bcl-2 were observed in arbutin- and acetylated arbutin-treated cells. Therefore, arbutin and acetylated arbutin were found to exert pro-apoptotic effects on B16 murine melanoma cells, mediated through the mitochondrial pathway. The findings of the present study also support the use of acetylated arbutin as a new potential candidate agent for skin whitening and melanoma treatment.
... Plasma-activated medium-regulated proliferation of melanocytes. Culture medium was exposed to NAP plasma for 4,8,12,16, and 20 min. Because the generation of radicals was proportional to the exposure duration to NAP plasma, the exposure duration of the culture medium to plasma corresponded to the PAM dose. ...
... Because the generation of radicals was proportional to the exposure duration to NAP plasma, the exposure duration of the culture medium to plasma corresponded to the PAM dose. To investigate the effect of PAM on their viability, melanocytes were exposed to PAM for 4,8,12,16, and 20 min, and subsequently incubated for 48 h. According to the tetrazolium (MTT) assay (Fig. S1), PAM seemed to have cytotoxic effects on melanocytes. ...
... α-MSH is a class of peptide hormones that are produced by cells in the intermediate lobe of the pituitary gland and that stimulate the production and release of melanin by melanocytes. Arbutin, which is one of the most widely prescribed skin-lightening and depigmenting agents worldwide, was used as a negative control 12 . The color of cell lysates dissolved in NaOH clearly showed the melanogenesis effect of PAM (Fig. 4A). ...
Article
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Non-thermal atmospheric pressure (NAP) plasma has demonstrated potential in biomedical applications, such as cancer treatment, bactericidal sterilization, and cell growth promotion or inhibition. In this study, for the first time, we demonstrated on–off switching of cell cycle progression and regulated melanogenesis in normal human skin melanocytes by NAP plasma-activated medium (PAM). The melanocytes were exposed to NAP plasma at durations varying from 0 to 20 min, and the effects of PAM on cell proliferation, cell cycle progression, and melanogenesis were investigated. Although PAM showed no cytotoxicity, the proliferation of melanocytes was inhibited. The melanocyte cell cycle was arrested by PAM for a relatively short period (48 h), after which it recovered slowly. PAM promoted melanogenesis through the activation of the enzymes tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. These effects seem to be related to reactive oxygen species induced by PAM. Our finding that PAM modulates the cell cycle may provide insight into the recurrence of cancer. The regulation of the melanogenesis of melanocytes may facilitate the control of skin tone without incurring negative side effects.
... In a recent study, human melanoma HMV-II cells were used as an alternative to B16 [6,7]. α-Arbutin (α-ARB), the epimer of β-ARB (l " Fig. 1), has inhibitory effects on melanogenesis and tyrosinase [8,9]. However, it remains unclear whether β-ARB or α-ARB has strong activity against HMV-II, and the inhibitory effects of KA on melanogenesis and tyrosinase using HMV-II are also unclear. ...
Article
The inhibitory effects of α-arbutin, β-arbutin, and kojic acid on melanogenesis, tyrosinase activity, and tyrosinase protein expression in mouse melanoma cells (B16-4A5) and human melanoma cells (HMV-II) were directly compared. β-Arbutin showed a stronger inhibitory effect on melanogenesis and tyrosinase expression in B16-4A5 cells than α-arbutin and kojic acid. Kojic acid showed a stronger inhibitory effect on mushroom and B16-4A5 tyrosinase activity than α-arbutin and β-arbutin. In contrast, kojic acid inhibited all of these effects more strongly than α-arbutin or β-arbutin in HMV-II cells. These results suggest that kojic acid may be used as a positive control for the inhibitory melanogenesis assay, and for tyrosinase activity and tyrosinase expression assays that use HMV-II cells. Moreover, using HMV-II cells with kojic acid as the positive control may facilitate the search for new skin-whitening agents using natural products and provide an alternative to the B16-4A5 assay.
... The 50% inhibitory concentration (IC 50 ) of α-arbutin on human tyrosinase is 2.0 mM, whereas that of natural arbutin is higher than 30 mM (Kazuhisha et al. 2007;Sugimoto et al. 2003). Inhibitory effects of α-arbutin on melanin biosynthesis were examined in cultured human melanoma cells and a human skin model, and the results showed that α-arbutin efficiently inhibited melanin synthesis without cytotoxicity effect (Kazuhisha et al. 2007;Sugimoto et al. 2004). Therefore, biosynthesis of α-arbutin has attracted increasing attention. ...
Article
Full-text available
Arbutin, a glucoside of hydroquinone, is used as a powerful skin lightening agent in the cosmeceutical industry because of its strong inhibitory effect on the human tyrosinase activity. It is a natural compound occurring in a number of plants, with a β-anomeric form of the glycoside bond between glucose and hydroquinone. α-Arbutin, which glycoside bond is generated with α-anomeric form, is the isomer of natural arbutin. α-Arbutin is generally produced by transglucosylation of hydroquinone by microbial glycosyltransferases. It is interesting that α-arbutin is found to be over 10 times more effective than arbutin, and thus biological production of α-arbutin attracts increasing attention. Seven different microbial enzymes have been identified to be able to produce α-arbutin, including α-amylase, sucrose phosphorlase, cyclodextrin glycosyltransferase, α-glucosidase, dextransucrase, amylosucrase, and sucrose isomerase. In this work, enzymatic and microbial production of α-arbutin is reviewed in detail.
... α-Arbutin, a glycosylated hydroquinone, is a prominent natural compound used as a skin-whitening agent due to its antimelanogenesis effect without any cytotoxicity [1]. It has been reported that the inhibitory function of α-arbutin against tyrosinase is 10 times greater than that of its isomer, β-arbutin [2]. Specifically, α-arbutin only inhibits mammalian tyrosinase, while β-arbutin reduces tyrosinase activity by both melanoma ...
Article
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α-Arbutin (4-hydroquinone-α-D-glucopyranoside), an effective skin-lightening agent due to its considerable inhibitory effect on human tyrosinase activity, is widely used in the pharmaceutical and cosmetic industries. Recently, α-arbutin was prepared through transglucosylation of hydroquinone using microbial glycosyltransferases as catalysts. However, the low yield and prolonged reaction time of the biotransformation process of α-arbutin production limited its industrial application. In this work, an amylosucrase (ASase) from Xanthomonas campestris pv. campestris str. ATCC 33913 (XcAS) was expressed efficiently in Escherichia coli JM109. The catalytic property of the purified XcAS for the synthesis of α-arbutin was tested. The recombinant strain was applied for highly efficient synthesis of α-arbutin using sucrose and hydroquinone as glucosyl donor and acceptor, respectively. By optimizing the biotransformation conditions and applying a fed-batch strategy, the final production yield and conversion rate of α-arbutin reached 60.9 g/L and 95.5%, respectively, which is the highest reported yield by engineered strains. Compared to the highest reported value (<1.4 g/L/h), our productivity (7.6 g/L/h) was improved more than five-fold. This work represents an efficient and rapid method for α-arbutin production with potential industrial applications.
... The tyrosinase enzyme is activated by forming chelates with vital copper ions and suppresses tautomerization from dopachrome, acting as a reducing agent in melanin intermediates, thus blocking oxidation chain reactions at various points from tyrosine/dopa to melanin. Inhibition of the formation of melanin with alpha arbutin can cause the skin to become brighter without any side effects [4][5][6][7]. Skin lightening cream is one form of the cosmetic preparation, is a mixture of chemicals or other ingredients with properties that can bleach black spots (brown) on the skin. The purpose of using it for a long time in order to eliminate or reduce hyperpigmentation of the skin [8]. ...
Article
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Objective: The objective of this research was to formulate and evaluate alpha arbutin skin lightening cream using polyacrylate base by cold process.Methods: Five formulas of alpha arbutin skin lightening cream were prepared with Polyacrylate Base with different polyacrylate concentrations as a cream base for each formula. The various concentrations of polyacrylate (2, 3, 4, 5, and 6%) was used as a cream base. The resulting preparation was then examined and observed for consistency, color, odor, and stability for 28 d of storage.Results: The phase separation test results stated that the five cream formulas did not experience phase separation in 2500, 3000, and 3750 rpm centrifugations so that it could be concluded that the five cream formulas were stable in storage at room temperature for at least 12 mo. From the physical evaluation of cream preparations, it was found that the cream formula with 6% polyacrylate concentration gave the best stability results. Cream safety testing results stated that the cream did not irritate the skin of the wearer.Conclusion: The results obtained in this research work clearly indicated alpha arbutin cream can be formulated using polyacrylate as a cream base, and in its manufacture, it can be carried out with a cold process.
... This has been demonstrated in cultured human melanoma cells. Its effects are further demonstrated by its effectiveness of inhibition of mushroom tyrosinase in vitro (75,76). ...
Article
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A blemish free, even-toned skin is universally associated with healthy skin. This reasoning makes people desire to have a flawless skin. Melanin is a naturally occurring pigment in humans. This pigment is responsible for skin, hair, and eye color, therefore determines our race and phenotypic appearance. On darker skin types, it is common that melanin production processes malfunctions. These malfunctions often lead to overproduction and secretion of melanin. As a result, unwanted pigmentary problems such melasma occur. Due to unknown etiology and its recurrence in nature, melasma is challenging to treat. The current available melasma treatment options often produce undesired side effects and suboptimum results. First-line topical treatments usually involve hydroquinone or topical steroids. Apart from the irritant reactions, this treatment mode is not suitable for all skin types. Skin care specialists are in search of an effective long-term cosmetics and cosmeceuticals to address hypermelanosis problems. Understanding of naturally occurring depigmenting agents provides an opportunity for more effective ways to manage melasma in all skin types. This review considers the benefits of naturally occurring ingredients which could help address skin pigmentation problems and broaden the choice for skin-lightening treatments.
... It acts by suppressing the tyrosinase enzyme present in the melanosomes, which boosts melanin production at non-cytotoxic concentrations other than reducing the synthesis and expression of such enzyme (Zhu & Gao, 2008). It is reported that arbutin's activity comes from its structural homologies with its substrate (tyrosine), which causes the competitive suppression of tyrosinase catalytic activity (Sugimoto et al., 2004). A number of studies have revealed that alpha-arbutin (a-arbutin) (or 4-hydroxyphenyl alpha-glucopyranoside) shows more substantial inhibitory potential on human tyrosinase, reaching ten times greater effect compared to beta-arbutin, which promotes its efficacy in attaining the required whitening effect on the skin (Yang et al., 2018). ...
... In the HMV-II human cell line, α-arbutin does not appear to inhibit cell viability below 1.0 mmol/L; however, the number of cells was significantly reduced at a concentration of 2.0 mmol/L. 24 ...
Article
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Introduction The development of tyrosinase inhibitors is a hot research topic. Recently, the Chinese herb Paeonia suffruticosa Andrews, commonly named as Cortex Moutan (CM), was reported as being capable of reducing melanogenesis. We developed an A2058 human melanoma cell model to test the safety and efficacy of tyrosinase inhibition. The aim was to further clarify the bioactivities of CM extracts and paeonol for the purpose of skin whitening. Methods The 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) scavenging activity, total polyphenol and flavonoid contents, and in vitro tyrosinase inhibitory effects of water and ethanol CM extracts were determined. Cellular inhibitions of tyrosinase and melanin production were also evaluated. Results Water and ethanol CM extracts were both shown to have strong DPPH scavenging abilities in a dose‐dependent manner. The polyphenol content was higher in the ethanol CM extract compared to the water extract, while the flavanone content was comparable. Kinetic analyses revealed that the ethanol CM extract and paeonol are noncompetitive tyrosinase inhibitors. The cellular melanin content and l‐DOPA oxidation assays demonstrated that the ethanol CM extract was an appropriate alternative whitening agent to paeonol and arbutin in ultraviolet‐induced A2058 human melanoma cells. Conclusion The results of this study suggest that a human cell model is more suitable for determining tyrosinase activity than mouse cell models for determining cellular tyrosinase activity and melanin production. The ethanol CM extract was also confirmed as a promising ingredient in sun protection and skin whitening cosmetics. Future work should focus on melanogenesis‐related gene expressions.
... Among these compounds, arbutin is produced biosynthetically from hydroquinone and glucose, and is present in pears (Pyrus serotina Rehder), Achillea millefolium, and a number of Ericaceae species. In addition, as arbutin is an inhibitor of tyrosinase at the post-translation level, it has a whitening effect on human skin without any side effects [1,2]. However, arbutin is significantly more expensive than other whitening compounds, and thus, its detection and isolation from natural sources are of particular importance. ...
Article
Arbutin is a skin-whitening agent found in plants such as the Ericaceae species. Herein, we report the development and validation of a method based on gas chromatography–mass spectrometry for the analysis of arbutin in plant extracts. Prior to analysis, silylation with trimethylsilyl (TMS) reagent produced 5TMS and 4TMS derivatives, for which the peak area ratios varied significantly prior to sample injection. However, the use of [d4]-arbutin as an internal standard overcame this issue, with little variation in the observed peak area ratios. Method validation confirmed the excellent linearity (r² = 0.9989), recovery (accuracy 101.4–105.7%), precision (relative standard deviation ≤ 3.43%), limit of detection (0.03 μg mL⁻¹), and limit of quantitation (0.08 μg mL⁻¹) of this novel method. Finally, our method was applied to the detection of arbutin in various plant species. To the best of our knowledge, arbutin was detected and measured in Prismatomeris tetrandra and Phyllanthus reticulatus samples for the first time. Graphical Abstract Open image in new window
... The other arbutin isomer is α-arbutin with α-anomeric glycoside bond. Both of isomers are commonly used in skin-whitening cosmetics and hyperpigmentation therapy (Migas and Krauze-Baranowska 2015), while α-arbutin displays ten times stronger inhibitory activity than β-arbutin (Sugimoto et al. 2004). With the rapid growth of global skin-whitening cosmetics market, the environmentfriendly production of α-arbutin attracts wide interests since it can be synthesized by one-step enzymatic glycosylation of HQ. ...
Article
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α-Arbutin is an effective skin-whitening cosmetic ingredient and hyperpigmentation therapy agent. It can be synthesized by one-step enzymatic glycosylation of hydroquinone (HQ), but limited by the low yield. Amylosucrase (Amy-1) from Xanthomonas campestris pv. campestris 8004 was recently identified with high HQ glycosylation activity. In this study, whole-cell transformation by Amy-1 was optimized and process scale-up was evaluated in 5000-L reactor. In comparison with purified Amy-1, whole-cell catalyst of recombinant E. coli displays better tolerance against inhibitors (oxidized products of HQ) and requires lower molar ratio of sucrose and HQ to reach high conversion rate (> 99%). Excess accumulation of glucose (0.6-1.0 M) derived from sucrose hydrolysis inhibits HQ glycosylation rate by 46-60%, which suggests the importance of balancing HQ glycosylation rate and sucrose hydrolysis rate by adjusting the activity of whole-cell catalyst and HQ-fed rate. Using optimal conditions, 540 mM of final concentration and 95% of molar conversion rate were obtained within 13-18 h in laboratory scale. For industrial scale-up production, 398 mM and 375 mM of final concentration with high conversion rates (~ 95%) were obtained in 3500-L and 4000-L of reaction volume, respectively. These yields and productivities (4.5-4.9 kg kL-1 h-1) were the highest by comparing to the best we known. Hence, high-yield production of α-arbutin by batch-feeding whole-cell biotransformation was successfully achieved in the 5000-L reaction scale.
... For example, hydroquinone, arbutin, kojic acid, and ellagic acid, which are used as typical skin-whitening agents, reduce the melanin content in cells or the skin of animals. Moreover, these compounds and ergothioneine inhibit tyrosinase activity (Jimbow et al. 1974;Battaini et al. 2000;Shimogaki et al. 2000;Sugimoto et al. 2004;Lajis et al. 2012;Liao et al. 2012). In particular, kojic acid and ellagic acid are predicted to competitively inhibit tyrosinase activity by chelating copper ions (Shimogaki et al. 2000;Battaini et al. 2000;Lima et al. 2014), suggesting that low molecular weight compounds reactive to metal ions may inhibit tyrosinase activity. ...
Article
The novel organic selenium compound, selenoneine, is found in the blood of tuna and has metal-binding activity. In this report, selenoneine displays tyrosinase inhibitory activity. When murine B16 melanoma cells were cultured in the presence of 1.0 μM selenoneine, the melanin content in the cells was reduced to 46.5% compared with the cell-induced melanin synthesis, and cellular tyrosinase activity was suppressed. In 3D-cultured human melanocytes, melanin accumulation was also decreased, to 39.7% and 23.0% by 1.0 and 5.0 μM selenoneine, respectively, compared with the control cells. Both cellular and purified enzyme assays showed that selenoneine inhibited tyrosinase activity against the substrate, l-3,4-dihydroxyphenylalanine (l-DOPA). An in silico docking simulation study supported a molecular mechanism in which selenoneine chelates copper ions in the active center of tyrosinase and prevents the reaction between tyrosinase and l-DOPA. These findings suggest that selenoneine has a novel biological function by inhibiting tyrosinase via copper chelation.
... Tyrosinase not only incorporates menalin production but also might be responsible for malignant cancer. Again, in whitening enhancement is due to the inhibition of tyrosinase activities (Sugimoto et al. 2004. Consequently, mushroom tyrosinase is popular among researchers due to its easy availability. ...
Article
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It was intended to investigate the biological activities of different solvent extracts of the fruiting bodies of Calocybe indica (P & C). The bioactive compounds were extracted using acetone, methanol, and hot water. The antioxidant activities of three different extracts at 0.5-20.0 mg/ml of C. indica on β-carotene-linoleic acid ranged from 65.83-92.56, 62.79-93.06 and 61.42-92.12%, respectively. The highest (2.825) reducing power inhibition was recorded in methanolic extract, while the lowest (2.332) was in hot water at the concentration of 8 mg/ml. The free radicle scavenging activity was more pronounced in methanolic extract. Chelating effect of C. indica was significantly strong as compared to positive control. The xanthine oxidase and tyrosinase inhibitory activity of the acetone, methanol, and hot water extracts of C. indica increased with increasing concentration. Thirteen phenolic compounds i.e. biochanin-A, caffeic acid, chlorogenic acid, ferulic acid, formononetin, gallic acid, hesperetin, homogentisic acid, naringenin, naringin, protocatechuic acid, resveratrol and vanillin were detected from acetonitrile-hydrochloric acid extract. The result indicated that the maximum and minimum concentrations were recorded for gallic acid (29 μg/g) and formononetin (10 μg/g). Thus, it could be suggested that C. indica has the potential to be used as antioxidant and tyrosinase protection system of the human body against oxidative damage and other complications.
... In addition to the phenolic compounds determined in our study, cranberry is also known to be rich in arbutin, a potent antioxidant with a hydroquinone moiety that inhibits the activity of tyrosinase and inhibits melanosome maturation [25]. The low IC 50 values of CP fractions are indicative of the presence of highly bioactive compounds against tyrosinase inhibition. ...
Article
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Background: Cranberry pomace (CP), an underutilized by-product from juice processing, contains a wide range of biologically active compounds that can be recovered and used in a variety of applications in functional foods and nutraceuticals. Methods: In this study, analytical chemical techniques such as solvent extractions and characterization of extracts in respect with their phenolic content were performed using ultra-high performance liquid chromatography mass spectrometry (UPLC-MS) and spectrophotometry. Crude CP extract and its phenolic acids, flavonols, anthocyanins and proanthocyanidins–rich fractions were then evaluated for their anti-oxidant capacity, tyrosinase inhibitory activity, and anti-proliferation activity against hepatocellular carcinoma HepG2 cells. Results: On a dry weight basis, the different CP fractions contained seven major anthocyanins (0.1-125 mg/g), six major phenolic acids (0.8-31 mg/g), seven flavonols (1-126 mg/g) and five flavan-3-ols (0.1-12 mg/g). Fractions rich in flavonols exhibited the most potent antioxidant capacities with ferric ion reducing antioxidant power values of 1.8-1.9 mmole/g and 2, 2-diphenyl-1-picrylhydrazyl radical scavenging IC50 values of 15.1-15.2 mg/L respectively. On the other hand, fractions rich in phenolic acids and flavan-3-ol monomers demonstrated the most potent anti-tyrosinase activity (IC50=6.1-6.2 mg/L) and anti-proliferative activity (IC50=7.8-15.8 mg/L). Generally, all the fractions exhibited a dose-response relationship in the selected biological activity assays. Conclusion: This study suggests an effective utilization of CP to obtain biologically active fractions with potential to be used in functional foods and nutraceuticals designed for the prevention of chronic diseases associated with oxidative stress.
... Various studies have demonstrated that arbutin is a safe and effective treatment for melisma. [10][11][12][13] Arbutin can be divided into two types; 1) α-arbutin (FIGURE 1) and 2) β-arbutin. With its dose-dependent action and less side effects than hydroquinone, α-arbutin is ten times more potent than β-arbutin. ...
Article
Concentrated 7% w/w a-arbutin cream was formulated and evaluated using O/W and W/O emulsion bases as an extemporaneous preparation for melasma treatment. Cream bases were formulated with two pH values, 4.0 and 5.5, using a hot process. The stability of the creams was studied for 60 days under three storage conditions (i.e., 2°C to 8°C, 30°C, 40°C). Cream characteristics and all aspects of product stability including physical, chemical, and microbial were investigated. Stability was defined as no dramatic change in color, viscosity, pH, and no visible microbial growth. For stability, at least 90% of the initial a-arbutin concentration quantified by stability-indicating high-performance liquid chromatography must be obtained. It was found that pH had no influence on the a-arbutin or formulations' stability. All formulations had a-arbutin remaining higher than 90% (approximately 92%) after being stored for 60 days in all storage conditions with no significant changes in pH or viscosity. All samples complied with the microbial limits test for nonsterile pharmaceutical preparation for cutaneous products. However, a color change was detected in O/W and W/O emulsions, especially at 40°C storage condition within 28 and 14 days, respectively. Drug crystals were observed in W/O emulsion stored at 2°C to 8°C. Concerning the in vitro drug release, a-arbutin was released from O/W emulsion but not from W/O emulsion. From the above results, the O/W emulsion that was developed in this study can be used as a cream base for concentrated a-arbutin as an extemporaneous preparation. The developed a-arbutin cream prepared using O/W emulsions can be used as an extemporaneous preparation with a beyond-use date of 60 days when stored at room temperature (30°C) and in the refrigerator (2°C to 8°C).
... cell-culture skin models, cadavers or animal skin; porcine, rabbit, rat) or artificial (e.g. liquid suspensions, gelatinous substances, elastomers, epoxy resins, textiles or metals) skin models available that could be used in many kinds of investigations, such as cosmetology, drug delivery, biology, and medicine, as well as ballistic, optical or thermal analysis [1,[6][7][8][9]. Among all possible materials, only a few can be considered to be skin models that mimic the frictional behaviour and friction-related properties of human skin [1,[10][11][12]. ...
Article
The properties of human skin strongly depend on hydration. Skin friction, elasticity and roughness change significantly in the presence of water. This paper presents a new bio-mimicking gelatine-based physical skin model that simulates the frictional behaviour of human skin against a widely-used standard textile under dry and wet conditions and over a broad range of applied normal load (0.5-5N) and amount of water at the interface (0–100 µl/cm²). The proposed skin model shows good agreement with the frictional behaviour of human skin both in dry and wet conditions. In addition, the tensile Young's modulus and surface roughness exhibit changes as a function of the amount of water that are similar to those of human skin. Potential applications of the model are in the testing and development of textile materials that mechanically interact with human skin.
... Melasma is one of the most common cosmetic disorders and has many adverse effects on the quality of life because its treatment is often lifelong and sometimes unsatisfactory (Crocco et al., 2015;Zhong et al., 2015). The topical use of tyrosinase inhibitors is recognized as the best therapy for treating melasma because tyrosinase is a rate-limiting enzyme found within melanocytes (Maeda and Fukuda, 1996;Sarkar et al., 2013;Sugimoto et al., 2004). However, most of the prescribed tyrosinase inhibitors that include hydroquinone, kojic acid, azelaic acid, vitamin C, and α-arbutin are hydrophilic in nature (Ogbechie-Godec and Elbuluk, 2017). ...
Article
The potential role of using carrageenan/locust bean gum (coded as κC: LBG) hydrogel to enhance transdermal delivery of hydrophilic actives was investigated in this study. To this purpose, κC:LBG hydrogel embedded with arbutin (coded as κC:LBG:AR) was investigated for their viscosity, physical-chemical, thermal and morphological properties, in vitro cytotoxicity, and release profile, and the in vivo performance was compared to arbutin cream. The κC:LBG:AR hydrogel showed viscosity values higher than those found in κC:LBG. Overall, both hydrogels demonstrated smooth surfaces and are cytocompatible in vitro. DSC showed that κC:LBG:AR hydrogel enhanced transdermal delivery through the skin by changing the structural organization of the lipid bilayer. The κC:LBG:AR hydrogel showed a significant increase in skin hydration and reduction in the melanin index as compared with commercial cream. These findings suggest that κC:LBG hydrogel possesses great potential to be used as a green platform for enhancing transdermal delivery.
... Therefore, tyrosinase inhibitors may be clinically helpful in dealing with skin cancer (Momtaz et al., 2008). Skin whitening is believed to be partly due to the inhibition of tyrosinase activity (Burdock et al., 2001;Hakozaki et al., 2002;Sugimoto et al., 2004). Irrespective of the medicinal importance or therapeutic potential of P. nebrodensis, there have not been many studies on the antioxidant and antityrosinase properties. ...
Article
Pleurotus nebrodensis has been widely used for nutritional and medicinal purposes. The aim of the present study was to evaluate the antioxidant activities and tyrosinase inhibitory effects on the fruiting bodies of P. nebrodensis extracted with acetone, methanol and hot water. The antioxidant activities were performed on ß-carotene-linoleic acid, reducing power, 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrous ions chelating abilities and xanthine oxidase inhibitory activity. In addition to this, phenolic compounds were also detected. The acetonic and methanolic extracts of P. nebrodensis showed the strongest ß-carotene-linoleic acid inhibition as compare to hot water extract. At 8 mg/ml, the acetonic extract showed a high reducing power of 1.86. The scavenging activity on DPPH radicals, the acetonic and methanolic extracts were effective than that of hot water extract. At lower concentrations, chelating effect of tested mushroom was significantly effective as compare to positive control. Gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid, naringenin, hesperetin, formononetin and biochanin-A were detected from acetonitrile and hydrochloric acid solvent extract. The xanthine oxidase and tyrosinase inhibitory activities of the acetonic, methanolic and hot water extracts increased with increase of concentration. This study suggests that fruiting bodies of P. nebrodensis can potentially be used as a readily accessible source of natural antioxidants.
... Arbutin, a naturally occurring glycoside of hydroquinone, acts as an inhibitor of the enzyme tyrosinase, which converts tyrosine into melanin in the skin [1,2]. For this reason, arbutin is widely used in cosmetics as the whitening agent for human skin. ...
Article
Arbutin is a very safe whitening agent for human skin. Since it is more expensive than other agents and has a challenging synthesis, novel methods to obtain this valuable agent are needed. In this study, we developed a precise and accurate method to detect and quantify arbutin using stable isotope dilution liquid chromatography–mass spectrometry (LC–MS). One challenge that needed to be overcome was the matrix effect occurring during the LC–MS analysis due to the analyte ionisation enhancement or suppression in the electrospray ionisation source by co-eluting compounds. Notably, arbutin had different matrix effects in the various sample matrices. A solution to this problem was the use of [d4]-arbutin as a stable isotope-labelled internal standard (SIL-IS), as it compensated the matrix effect of arbutin because it was affected by almost the same matrix effect. The validation of the developed method showed excellent linearity (r² = 1.000), precision (relative standard deviation ≤ 2.5%), accuracy (recovery, 97.42–98.52%), limit of detection (0.03 μg/mL), and limit of quantification (0.1 μg/mL). Finally, the method of arbutin detection was applied to blueberry leaves to compare the precision and accuracy results obtained by performing stable isotope dilution using LC–MS and gas chromatography–mass spectrometry. The method was applied to strawberry leaves and pear peels, indicating that the SIL-IS method can be expected to find application in the arbutin analysis in other plants.
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The skin is commonly exposed to pharmaceutical active ingredients in the form of topically applied therapeutics or systemically exposed following oral, intravenous, or inhaled therapeutics administration. Cutaneous reactions are among the most prevalent adverse effects of therapeutic drugs. This chapter covers the state-of-the-art of in vitro human skin models and the utility of these models for preclinical drug development applications including screening for skin sensitization, phototoxicity, irritation, genotoxicity, pigmentation effects, and percutaneous absorption. 3D RhE models may also be useful for in vitro evaluation of percutanous absorption. A number of in vitro models have been developed for assessing the phototoxic potential of chemicals. Some of these in vitro skin model systems have been adopted by regulatory authorities and have been incorporated into regulatory guidelines. The most widely used in vitro phototoxicity assay is the in vitro 3 T3 Neutral Red Uptake Phototoxicity Test (3 T3 NRU PT).
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The known derivatives from hydroquinone, α and β-arbutin, are used as depigmenting agents. In this work, we demonstrate that the oxy form of tyrosinase (oxytyrosinase) hydroxylates α and β-arbutin in ortho position of the phenolic hydroxyl group, giving rise to a complex formed by met-tyrosinase with the hydroxylated α or β-arbutin. This complex could evolve in two ways: by oxidizing the originated o-diphenol to o-quinone and deoxy-tyrosinase, or by delivering the o-diphenol and met-tyrosinase to the medium, which would produce the self-activation of the system. Note that the quinones generated in both cases are unstable, so the catalysis cannot be studied quantitatively. However, if 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate is used, the o-quinone is attacked, so that it becomes an adduct, which can be oxidized by another molecule of o-quinone, generating o-diphenol in the medium. In this way, the system reaches the steady state and originates a chromophore, which, in turn, has a high absorptivity in the visible spectrum. This reaction allowed us to characterize α and β-arbutin kinetically as substrates of tyrosinase for the first time, obtaining a Michaelis constant values of 6.5 ± 0.58 mM and 3 ± 0.19 mM, respectively. The data agree with those from docking studies that showed that the enzyme has a higher affinity for β-arbutin. Moreover, the catalytic constants obtained by the kinetic studies (catalytic constant = 4.43 ± 0.33 s⁻¹ and 3.7 ± 0.29 s⁻¹ for α and β-arbutin respectively) agree with our forecast based on 13 C NMR considerations. This kinetic characterization of α and β-arbutin as substrates of tyrosinase should be taken into account to explain possible adverse effects of these compounds.
Article
Phenolic compounds are an important class of chemicals with various beneficial bioactivities. However, they are usually poorly soluble in water and unstable while some of them are toxic. Glycosylation can significantly improve the solubility, stability, and bioactivity of phenolic compounds. The enzymatic method for glycosylation can form a specific glycosidic bond in one step and under environment-friendly conditions. This review covers the progress made in the application of two classes of enzymes, namely, glycosyltransferases and glycosidases, for the glycosylation of phenolic compounds, and illustrates the impact of glycosylation on the properties of these compounds.Recent progress in the enzymatic glycosylation of phenolic compoundsAll authorsLijuan Xua, Tingting Qia, Li Xua, Lili Lua* & Min Xiaob*DOI:http://dx.doi.org/10.1080/07328303.2015.1137580Published online:24 March 2016PowerPoint slideOriginal jpg (31.00KB)Display full size
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Endothelin-1 (ET.1), a powerful vasoconstrictor and mitogen tor melanocytes, might be responsible for pigmentation that occurs after ultraviolet (UVB) -irradiation. We have previously reported that Sechium edule (5. edule) extract reduces ET-1 secretion in cultured human keratinocytes following UVB irradiation. In this study, we investigated the effect of S. edule extract on melanin synthesis and analyzed the polyphenols contained in the extract. A three-dimensional human skin model comprising normal epidermal keratinocytes and melanocytes was subjected to macroscopical pigmentation and quantitative analysis. The S. edule extract showed an inhibitory effect on macroscopic and microscopic darkening and melanin content; however it did not show an inhibitory effect on tyrosinase enzyme activity. High-performance liquid chromatography with coulometric array detection was employed to determine the polyphenols contained in the extract. The luteolin, ferulic acid, p-coumaric acid, vanillic acid, protocatechuic acid and p-hydroxybenzoic acid were found to be 6.6 μg/g, 5.6 /μ/g, 3.3 μg/g, 6.1 μg/g, 1.5 μg/g and 0.8 μg/g (dry weight), respectively. This polyphenolic complex might result in the inhibitory effect of melanin synthesis.
Article
We found a new reaction of sucrose phosphorylases; transglycosylation of carboxyl group. Sucrose phosphorylases from two different sources were tested for glycosylation of carboxylic acid compounds. Streptococcus mutans sucrose phosphorylase showed remarkable transglycosylating activity, especially under acidic conditions. Leuconostoc mesenteroides sucrose phosphorylase exhibited very weak transglycosylating activity. When benzoic acid and sucrose were used as an acceptor and a donor molecule, 1-. O- benzoyl αD-glucopyranoside was produced which was identified by 1D- and 2D-NMR analyses of the purified product and its acetylated product. S. mutans sucrose phosphorylase showed broad acceptor-specificity. The sucrose phosphorylase catalyzed transglycosylation to various carboxylic compounds such as short-chain fatty acids, hydroxy acids, dicarboxylic acids, phenolic carboxylic acids, and acetic acid.
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The cultured medium of mesenchymal stem cell is a valuable resource for a variety of stem cell-derived cytokines and growth factors with a potential therapeutic effect on skin diseases. Here, we investigated the effect of the cultured medium of human umbilical cord blood-derived mesenchymal stem cell (CM-hCBSC) on melanogenesis in mouse B16 melanoma cells stimulated by α-melanocyte stimulating hormone (α-MSH). Our results show that CM-hCBSC inhibits melanin synthesis due to the decrease in cellular tyrosinase activity in B16 melanoma cells without cytotoxicity. The activity of melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 were evaluated by western blot and semi-quantitative RT-PCR. The CM-hCBSC down-regulated the protein and mRNA expression levels of tyrosinase and TRP-1 in a dose-dependent manner. In addition, TRP-2 protein and mRNA expression levels were found to be down-regulated to a lesser extent. We also measured the level of cytokines in CM-hCBSC using the Luminex total system. It was found that the top enriched cytokine in the CMhCBSC was interukine-6 (IL-6), known as a potent regulator of melanin synthesis together with IL-1α and tumor necrosis factor α (TNF- α). These results indicate that secretary factors including IL-6 in the CM-hCBSC may inhibit melanin synthesis by down-regulating the expression of tyrosinase and TRP1. This study suggests that CMhCBSC has a therapeutic effect as an anti-melanogenic agent and may be effective against hyperpigmentation disorders. © 2014, The Korean Tissue Engineering and Regenerative Medicine Society and Springer Science+Business Media Dordrecht.
Article
The application of transdermal delivery to a wider range of drugs is limited due to the significant barrier to penetration across the skin which is associated with the stratum corneum layer of the epidermis. In previous study in the literature, the feasibility and effects of the ultrasound (US) contrast agent, microbubbles (MBs) as the penetration enhancers for transdermal delivery in vivo were firstly demonstrated. In this study, the penetration depth, concentration, and efficiency of transdermal α-Arbutin delivery after MBs treatment with US in mice were demonstrated for 4 weeks. The penetration of α-arbutin on skin was enhanced by using ultrasound energy and MBs either for in vitro or for in vivo experiments. Experiment parameters were randomly divided into four groups (n=5 animals per group): (1) only penetrating α-Arbutin (C); (2) US combines with penetrating α-arbutin (U) (3) US combines with MBs contrast agent and penetrating α-arbutin (UB); (4) US combines with diluted MBs and penetrating α-arbutin (UBD). According to the results, the penetration depth of agarose phantom and pigskin of UBD group increase 47% and 84%, respectively. The in vitro skin permeation of 2% α-arbutin, UBD group was 83% greater than control group after 3 hour of permeation study. For in vivo study, the whitening effect (luminosity index) of mice skin in UBD group significantly increase 25% in one week, 34% in two weeks and tends towards stability in three weeks (37%) in C57BL/6J mice over a 4-week experimental period. Our results investigated that the treatments of ultrasound and MBs can increase skin permeability, enhance α-arbutin delivery to inhibit melanogenesis and not damage the skin in mice.
Article
Discarded branch prunings from Japanese pear trees (Pyrus pyrifolia cv. Kousui) are promising resources containing polysaccharides and polyphenols. This study aimed at the total utilization of discarded branches. Arbutin is an inhibitor of biosynthesis of the human pigment melanin and extensively used as a human skin-whitening agent. It was found that most of the arbutin in the branches was contained in the bark (27.1 mg/g dry matter) compared with that in wood (5.2 mg/g dry matter). We evaluated the effect of the extracted solution containing arbutin (ESCA) from the bark on melanin formation in B16 melanoma cells. At a concentration of 4.0 × 10−3 % of ESCA, treated B16 melanoma cells showed 77.9 ± 3.9 % of the melanin production of untreated cells, with no cytotoxicity. The residue (wood) after arbutin extraction contained abundant holocellulose (69.8 %), and was accordingly hydrolyzed with cellulase and then converted into ethanol by Saccharomyces cerevisiae BA11, after microwave irradiation pretreatment. In total, 0.79 g of arbutin and 8.84 g of ethanol were produced from the 100 g of dry branches.
Article
The present study was the first evaluation of the arbutin content, antioxidant activity, and whitening function of the unripe pears of five major Korean pear cultivars. Unripe pears were investiga-ted 30 days after florescence for possible utilization as a whitening ingredient, instead of being thrown away for thinning out. Among the five cultivars tested, Gamcheonbae and Manpungbae had significantly higher total phenolics and arbutin contents, while Niitaka had lower values of both total phenolics and arbutin. For whitening activity related to tyrosinase and cellular melanin formation, Manpungbae also showed the strongest tyrosinase inhibition (4.9%), and achieved 74% reduction of the cellular melanin compared to non-treated cells. These results indicate that unripe pears, especially the Manpungbae cultivar, could be useful for application as a possible natural whitening additive with high arbutin content and excellent whitening activity.
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To investigate the use of chitosan nanoparticles (CS-TPP-NPs) as carriers for α- and β-arbutin. In this study, CS-TPP-NPs containing α- and β-arbutin were prepared via the ionic cross-linking of CS and TPP and characterized for particle size, zeta potential, and dispersity index. The entrapment efficiency and loading capacity of various β-arbutin concentrations (0.1, 0.2, 0.4, 0.5, and 0.6%) were also investigated. SEM, TEM FTIR, DSC and TGA analyses of the nanoparticles were performed to further characterize the nanoparticles. Finally, stability and release studies were undertaken to ascertain further the suitability of the nanoparticles as a carrier system for α- and β-arbutin. Data obtained clearly indicates the potential for use of CS-TPP-NPs as a carrier for the delivery of α- and β-arbutin. The size obtained for the alpha nanoparticles (α-arbutin CSNPs) ranges from 147 to 274 d.nm, with an increase in size with increasing alpha arbutin concentration. β-arbutin nanoparticles (β-arbutin CSNPs) size range was from 211.1 to 284 dn.m. PdI for all nanoparticles remained between 0.2–0.3 while the zeta potential was between 41.6–52.1 mV. The optimum encapsulation efficiency and loading capacity for 0.4% α-arbutin CSNPs were 71 and 77%, respectively. As for β-arbutin, CSNP optimum encapsulation efficiency and loading capacity for 0.4% concentration were 68 and 74%, respectively. Scanning electron microscopy for α-arbutin CSNPs showed a more spherical shape compared to β-arbutin CSNPs where rod-shaped particles were observed. However, under transmission electron microscopy, the shapes of both α- and β-arbutin CSNP nanoparticles were spherical. The crystal phase identification of the studied samples was carried out using X-ray diffraction (XRD), and the XRD of both α and β-arbutin CSNPs showed to be more crystalline in comparison to their free form. FTIR spectra showed intense characteristic peaks of chitosan appearing at 3438.3 cm⁻¹ (-OH stretching), 2912 cm⁻¹ (-CH stretching), represented 1598.01 cm⁻¹ (-NH2) for both nanoparticles. Stability studies conducted for 90 days revealed that both α- and β-arbutin CSNPs were stable in solution. Finally, release studies of both α- and β-arbutin CSNPs showed a significantly higher percentage release in comparison to α- and β-arbutin in their free form. Chitosan nanoparticles demonstrate considerable promise as a carrier system for α- and β-arbutin, the use of which is anticipated to improve delivery of arbutin through the skin, in order to improve its efficacy as a whitening agent.
Article
Cosmetic dermatology preparations such as bleaching agents are ingredients with skin‐related biological activities for increasing and improving skin beauty. The possibility of controlling skin hyperpigmentation disorders is one of the most important research goals in cosmetic preparations. Recently, cosmetics containing herbal and botanical ingredients have attracted many interests for consumers of cosmetic products because these preparations are found safer than other preparations with synthetic components. However, high‐quality trial studies in larger samples are needed to confirm safety and clinical efficacy of phytotherapeutic agents with high therapeutic index. Arbutin (p‐hydroxyphenyl‐β‐d‐glucopyranoside) is a bioactive hydrophilic polyphenol with two isomers including alpha‐arbutin (4‐hydroxyphenyl‐α‐glucopyranoside) and β‐arbutin (4‐hydroxyphenyl‐β‐glucopyranoside). It is used as a medicinal plant in phytopharmacy. Studies have shown that alpha‐arbutin is 10 times more effective than natural arbutin. A comparison of IC50 values showed that α‐arbutin (with concentration 2.0 mM) has a more potent inhibitory activity on human tyrosinase against natural arbutin (with higher concentration than 30 mM). A review of recent studies showed that arbutin could be beneficial in treatment of various diseases such as hyperpigmentation disorders, types of cancers, central nervous system disorders, osteoporosis, diabetes, etc. This study was designed to describe the therapeutic efficiencies of arbutin.
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Kojic acid, β-arbutin, α-arbutin, and deoxyarbutin have been reported as tyrosinase inhibitors in many articles, but some contradictions exist in their differing results. In order to provide some explanations for these contradictions and to find the most suitable compound as a positive control for screening potential tyrosinase inhibitors, the activity and inhibition type of the aforementioned compounds on monophenolase and diphenolase of mushroom tyrosinase (MTYR) were studied. Their effects on B16F10 cells melanin content, tyrosinase (BTYR) activity, and cell viability were also exposed. Results indicated that α-arbutin competitively inhibited monophenolase activity, whereas they uncompetitively activated diphenolase activity of MTYR. β-arbutin noncompetitively and competitively inhibited monophenolase activity at high molarity (4000 µM) and moderate molarity (250–1000 µM) respectively, whereas it activated the diphenolase activity of MTYR. Deoxyarbutin competitively inhibited diphenolase activity, but could not inhibit monophenolase activity and only extended the lag time. Kojic acid competitively inhibited monophenolase activity and competitive–noncompetitive mixed-type inhibited diphenolase activity of MTYR. In a cellular experiment, deoxyarbutin effectively inhibited BTYR activity and reduced melanin content, but it also potently decreased cell viability. α-arbutin and β-arbutin dose-dependently inhibited BTYR activity, reduced melanin content, and increased cell viability. Kojic acid did not affect cell viability at 43.8–700 µM, but inhibited BTYR activity and reduced melanin content in a dose-dependent manner. Therefore, kojic acid was considered as the most suitable positive control among these four compounds, because it could inhibit both monophenolase and diphenolase activity of MTYR and reduce intercellular melanin content by inhibiting BTYR activity without cytotoxicity. Some explanations for the contradictions in the reported articles were provided.
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Endophytic fungi have been recognized as prolific producers of chemically diverse secondary metabolites. In this work, we describe a new representative of the order Helotiales isolated from the medicinal plant Bergenia pacumbis . Several bioactive secondary metabolites were produced by this Helotiales sp. BL 73 isolate grown on rice medium, including cochlioquinones and isofusidienols. Sequencing and analysis of the approx. 59 Mb genome revealed at least 77 secondary metabolite biosynthesis gene clusters, several of which could be associated with detected compounds or linked to previously reported molecules. Four terpene synthase genes identified in the BL73 genome were codon-optimized and expressed, together with farnesyl-, geranyl- and geranylgeranyl-pyrophosphate synthases, in Streptomyces spp. Analysis of recombinant strains revealed production of linalool and its oxidized form, terpenoids typically associated with plants, as well as a yet unidentified terpenoid. This study demonstrates the importance of a complex approach to the investigation of the biosynthetic potential of endophytic fungi using both conventional methods and genome mining. I mportance Endophytic fungi represent as yet underexplored source of secondary metabolites, some of which may have industrial and medical applications. We isolated a slow-growing fungus belonging to the order Helotiales from the traditional medicinal plant Bergenia pacumbis and characterized its potential to biosynthesize secondary metabolites. We used both cultivation of the isolate with subsequent analysis of compounds produced, bioinformatics-based mining of the genome, and heterologous expression of several terpene synthase genes. Our study revealed enormous potential of this Helotiales isolate to produce structurally diverse natural products, including polyketides, non-ribosomally synthesized peptides, terpenoids and RiPPs. Identification of meroterpenoids and xanthones, along with establishing a link between these molecules and their putative biosynthetic genes sets a stage for investigation of the respective biosynthetic pathways. Heterologous production of terpenoids suggests that this approach can be used for the discovery of new compounds belonging to this chemical class using Streptomyces bacteria as hosts.
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Polyphenols exhibit various beneficial biological activities and represent very promising candidates as active compounds for food industry. However, the low solubility, poor stability and low bioavailability of polyphenols have severely limited their industrial applications. Enzymatic glycosylation is an effective way to improve the physicochemical properties of polyphenols. As efficient transglucosidases, glycoside hydrolase family 70 (GH70) glucansucrases naturally catalyze the synthesis of polysaccharides and oligosaccharides from sucrose. Notably, GH70 glucansucrases show broad acceptor substrate promiscuity and catalyze the glucosylation of a wide range of non-carbohydrate hydroxyl group-containing molecules, including benzenediol, phenolic acids, flavonoids and steviol glycosides. Branching sucrase enzymes, a newly established subfamily of GH70, are shown to possess a broader acceptor substrate binding pocket that acts efficiently for glucosylation of larger size polyphenols such as flavonoids. Here we present a comprehensive review of glucosylation of polyphenols using GH70 glucansucrase and branching sucrases. Their catalytic efficiency, the regioselectivity of glucosylation and the structure of generated products are described for these reactions. Moreover, enzyme engineering is effective for improving their catalytic efficiency and product specificity. The combined information provides novel insights on the glucosylation of polyphenols by GH70 glucansucrases and branching sucrases, and may promote their applications.
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Uva-ursi (Arctostaphylos uva-ursi) is a shrub whose leaves are used to make medicine. Clinical trials are almost non-existent for this herb and indicate that uva-ursi may be beneficial for prevention of recurrent urinary tract infections but may not be effective for acute urinary tract infections. Uva-ursi has been reported to show antibacterial efficacy in more than 70 strains of bacteria known to exist in the urinary tract. Arbutin, the chief constituent found in the leaves of the herb, has been shown to have antibacterial properties via its metabolite hydroquinone glucuronide. The presence of hydroquinone glucuronide in the bladder prevents bacteria from adhering to the uroepithelium. This chapter examines some of the scientific research conducted on uva-ursi, both alone and in combination formulas, for treating cystitis and recurrent urinary tract infection. Finally, the chapter presents a list of uva-ursi’s active constituents, different Commonly Used Preparations and Dosage, and a section on “Safety and Precaution” that examines side effects, toxicity, and disease and drug interactions.
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Melanin pigmentation in the human skin results from complicated cellular mechanisms that remain to be entirely understood. Uneven melanin pigmentation has been counteracted by inhibiting synthesis or transfer of melanin in the skin. Recently, an enzymatic approach has been proposed, wherein the melanin in the skin is decolorized using lignin peroxidase. However, not many enzymes are available for decolorizing melanin; the most studied one is lignin peroxidase derived from a lignin degrading fungus, Phanerochaete chrysosporium. Our current study reveals that versatile peroxidase from Bjerkandera adusta can decolorize synthetic melanin. Melanin decolorization was found to be dependent on veratryl alcohol and hydrogen peroxide, but not on Mn2+. The degree of decolorization reached over 40% in 10 min at 37 °C and a pH of 4.5. Optimized storage conditions were slightly different from those for the reaction; crude enzyme preparation was the most stable at 25 °C at pH 5.5. Since the enzyme rapidly lost its activity at 50 °C, stabilizers were screened. As a result, glycerol, a major component in several cosmetic formulations, was found to be a promising excipient. Our results suggest that B. adusta versatile peroxidase can be considered for future cosmetic applications aimed at melanin decolorization.
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Arbutin is a compound of hydroquinone and D-glucose, and it has been over 30 years since there have been serious studies on the skin lightening action of this substance. In the meantime, there have been debates and validation studies about the mechanism of action of this substance as well as its skin lightening efficacy and safety. Several analogs or derivatives of arbutin have been developed and studied for their melanin synthesis inhibitory action. Formulations have been developed to improve the stability, transdermal delivery, and release of arbutin, and device usage to promote skin absorption has been developed. Substances that inhibit melanin synthesis synergistically with arbutin have been explored. The skin lightening efficacy of arbutin alone or in combination with other active ingredients has been clinically evaluated. Combined therapy with arbutin and laser could give enhanced depigmenting efficacy. The use of arbutin causes dermatitis rarely, and caution is recommended for the use of arbutin-containing products, especially from the viewpoint that hydroquinone may be generated during product use. Studies on the antioxidant properties of arbutin are emerging, and these antioxidant properties are proposed to contribute to the skin depigmenting action of arbutin. It is hoped that this review will help to understand the pros and cons of arbutin as a cosmetic ingredient, and will lead to future research directions for developing advanced skin lightening and protecting cosmetic products.
Article
Arbutin is a naturally occurring glycosylated product of hydroquinone. With the ability to interrupt melanin biosynthesis in epidermal cells, it is a promising cosmetic ingredient. In this study, a novel amylosucrase, Asmet, identified from a thermal spring metagenome, has been characterized for arbutin biosynthesis. Asmet was able to catalyze transglucosylation of hydroquinone to arbutin, taking sucrose as glycosyl donor, in the temperature range of 20 °C to 40 °C and pH 5.0 to 6.0, with the relative activity of 80% or more. The presence of chloride salts of Li, K, and Na at 1 mM concentration did not exhibit any notable effect on the enzyme's activity, unlike Cu, Ni, and Mn, which were observed to be detrimental. The hydroquinone (20 mM) to sucrose ratio of 1:1 to 1:10 was appropriate for the catalytic biosynthesis of arbutin. The maximum hydroquinone to arbutin conversion of 70% was obtained in 24 h of Asmet led catalysis, at 30 °C and pH 6.0. Arbutin production was also demonstrated using low-cost feedstock, table sugar, muscovado, and sweet sorghum stalk extract, as a replacement for sucrose. Whole-cell catalysis of hydroquinone to arbutin transglucosylation was also established.
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As a safe substitute for hydroquinone, β-arbutin, a natural plant substance, and its synthetic counterpart, α-arbutin, are used in depigmentation formulations. However, there are debatable points regarding the impact of arbutin on tyrosinase and the pigmentation process. To shed light on this issue, the effects of Pyrus biossieriana leaves extract (PbLE) and β-arbutin, extracted from PbLE, on mushroom tyrosinase (MT) were comprehensively examined. The study was focused on cresolase activity as the characteristic reaction of a tyrosinase. Kinetics studies disclosed that β-arbutin can modulate MT monophenolase activity from inhibition to activation or vice versa. β-Arbutin inhibited l-tyrosine (LTy) oxidation at concentrations < 0.3 mM but it increased (more than 400%) the enzymatic oxidation of l-tyrosine at the concentrations > 0.3 mM. An opposite pattern (activation then inhibition) was observed when a synthetic substrate was used instead of LTy. Computational studies, focused on the heavy chain of MT, indicated that β-arbutin effect could be overruled by the enzyme's ability to provide the ligand with a non-specific binding site (MTPc). A plausible mechanism was presented to show the influence of MTPc on the substrate pose in the active site. The possible determinant correlation between the findings of this research and the current studies on human tyrosinase role in the pigmentation process has been presented.
Article
α-Arbutin is an effective skin-whitening cosmetic ingredient and can be synthesized through hydroquinone glycosylation. In this study, amylosucrase (Amy-1) from Xanthomonas campestris pv. campestris 8004 was newly identified as a sucrose-utilizing glycosylating hydroquinone enzyme. Its kinetic parameters showed a seven-time higher affinity to hydroquinone than maltose-utilizing α-glycosidase. The glycosylation of HQ can be quickly achieved with over 99% conversion when a high molar ratio of glycoside donor to acceptor (80:1) was used. A batch-feeding catalysis method was designed to eliminate HQ inhibition with high productivity (> 36.4 mM h−1). Besides, to eliminate the serious inhibition caused by the accumulated hydroquinone oxidation products, the whole-cell catalysis was further proposed. 306 mM of α-arbutin was finally achieved with 95% molar conversion rate within 15 h. Hence, the batch-feeding whole-cell biocatalysis by Amy-1 is a promising technology for α-arbutin production with enhanced yield and molar conversion rate.
Article
Chitosan-coated magnetic nanoparticles ([email protected]) which used as substrate immobilization carrier of glucosyl acceptor for α-arbutin production is introduced in this study. Fe3O4@CTS microspheres were prepared via an oxidation hydrothermal method, and further modified with chitosan. Substrate hydroquinone (HQ) was immobilized on the Fe3O4@CTS. When 1.0 g/L Fe3O4@CTS was shaken in HQ solution (7.0 g/L) at 28 °C with 120 rpm for 12 h, the maximum amount of HQ immobilization on the Fe3O4@CTS could reach 312 mg HQ per gram Fe3O4@CTS. The optimal reaction conditions for α-arbutin synthesis were also investigated. When 25 g/L Fe3O4@[email protected] (70.9 mM HQ) was shaken in fermentation broth at 30 °C with 180 rpm for 40 h, the final yield of α-arbutin reached 7.85 g/L. This work is useful for the industrial-scale production of α-arbutin.
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The in vitro biochemical evaluation of the applicability of polymers carrying active substances (micelles and conjugates) was carried out. Previously designed amphiphilic graft copolymers with retinol or 4-n-butylresorcinol functionalized polymethacrylate backbone and poly(ethylene glycol) (PEG) side chains that included Janus-type heterografted copolymers containing both PEG and poly(ε-caprolactone) (PCL) side chains were applied as micellar carriers. The polymer self-assemblies were convenient to encapsulate arbutin (ARB) as the selected active substances. Moreover, the conjugates of PEG graft copolymers with ferulic acid (FA) or lipoic acid (LA) were also investigated. The permeability of released active substances through a membrane mimicking skin was evaluated by conducting transdermal tests in Franz diffusion cells. The biological response to new carriers with active substances was tested across cell lines, including normal human dermal fibroblasts (NHDF), human epidermal keratinocyte (HaCaT), as well as cancer melanoma (Me45) and metastatic human melanoma (451-Lu), for comparison. These polymer systems were safe and non-cytotoxic at the tested concentrations for healthy skin cell lines according to the MTT test. Cytometric evaluation of cell cycles as well as cell death defined by Annexin-V apoptosis assays and senescence tests showed no significant changes under action of the delivery systems, as compared to the control cells. In vitro tests confirmed the biochemical potential of these antioxidant carriers as beneficial components in cosmetic products, especially applied in the form of masks and eye pads.
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Alpha-arbutin (4-hydroxyphenyl alpha-glucopyranoside) is a known inhibitor of tyrosinase in keratinocytes; however, its effect on other genes and pathways in other skin cells has not been thoroughly investigated. In this study, we investigate the mechanism of alpha-arbutin activity in human dermal fibroblast cultures for 48 h. Results showed that the oxidative stress pathway was activated as alpha-arbutin reduced reactive oxygen species. In addition, we found a high possibility of wound healing and the upregulation of the insulin-like growth factor 1 receptor (IFG1R) pathway. We also investigated the role of the NRF2 gene in mediating the alpha-arbutin response. In silico comparative genomics analysis conducted using our original tool, SHOE, suggested transcription factors with a role in tumor suppression and toxicity response as candidates for regulating the alpha-arbutin-mediated pathway.
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Background/objectives: The picosecond Alexandrite laser was studied in our practice with the diffractive lens array and the flat optic to treat melasma. Methods and materials: Sixty patients with melasma were treated in a prospective investigation with the picosecond Alexandrite laser. Nineteen patients were treated with the flat optic and 41 patients were treated with the diffractive lens array. Treatments were performed with 1 pass at 2-week intervals for 6 treatments. The Melasma Severity Index (MSI) was used to evaluate the patients before treatment and 3 and 6 months after the final treatment session. Results: At 6 months after the last treatment, there was an 18.5% difference between the groups with a 75.7% improvement in the MSI in patients with the diffractive lens array and a 57.2% improvement in the MSI score in patients with the flat optic. At 6 months, there was recurrence of melasma in 5% of the cases with no hyperpigmentation with the diffractive optic in contrast to recurrence in 16% of the cases in the flat optic group and a transient macular hyperpigmentation in 21% of the cases. Conclusion: This investigation highlights the utility of a picosecond Alexandrite laser with a flat and diffractive lens to successfully treat a large percentage of Asian patients in a sunny climate.
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Since melanocytes are the origin of melanoma and some skin disorders such as melasma, they are important cells from the perspective of medicinal chemistry. Therefore, a medication that can simultaneously overcome these diseases will be a successful potential therapeutic agent. Arbutin with phenolic structure is a powerful natural anti-tyrosinase agent. Hence, the phenolic structure of this drug, prompted us to design its novel calix [4]arene-based cluster. Therefore, the present study reports the synthesis and in-vitro bio-activities of cyclic tetramer of arbutin in comparison to its simple drug unit as the reference medication. The in-vitro biological results showed amplified anti-tyrosinase (6-fold) and anti-melanoma (27-fold) activities, in addition to more aqueous solubility (8-fold) for this cluster in relation to arbutin. Therefore, compared to arbutin, more bioactive cluster can be considered as a novel water-soluble melanogenesis inhibitor with high anti-melanoma activity.
Article
Background Melanin plays an important role in protecting the skin against the harmful effects of solar radiation, but its abnormal accumulation may become an aesthetic problem, such as melasma and age spots. Aims The aim of this study was to evaluate the antiangiogenic and whitening effects of a depigmentation formulation (BLTX) using an in vitro model of human cell and skin culture. Methods Human fibroblasts, keratinocytes or melanocytes were treated with BLTX and subjected to oxidative stress by UV radiation or inflammatory stress with IL‐1α for quantification of melanin, tyrosinase, endothelin‐1, PAR‐2, VEGF and iNOS. Fragments of human skin, from elective plastic surgery, were treated with BLTX and subjected to histological evaluation with hematoxylin/eosin associated with Fontana‐Masson technique for melanin view. A parametric method, the one‐way analysis of variance (ANOVA) followed by the Bonferroni test, was used to compare data among all groups. Results The results demonstrated that BLTX promotes a reduction in VEGF and iNOS protein synthesis in cultured dermal fibroblasts, indicating an antiangiogenic property. In relation to whitening effect, BLTX was able to reduce the production of melanin in both systems, melanocytes and human skin cultures. The depigmenting action was also revealed by decreasing the levels of endothelin‐1, PAR‐2 and activity of tyrosinase, when compared to cultures exposed to UV radiation. Conclusion The results allow us to infer that BLTX presents an antiangiogenic effect indicating a role in the vascular component of melasma. Furthermore, the whitening property observed reinforces its use in the prevention and treatment of melasma.
Article
This article reviews the current status of skin tissue equivalents that have emerged as relevant tools in commercial and therapeutic product development applications. Due to rise of animal welfare concerns, numerous companies have designed skin model alternatives to assess the efficacy of pharmaceutical, skincare and cosmetic products in an in vitro setting, decreasing the dependency on such methods. Several validated test methods are performed on such models to investigate various characteristics including skin irritation, permeation, corrosion and hydration, genotoxicity and absorption. Skin models have also made an impact in determining the root causes of skin diseases and providing a means for developing an effective treatment in certain clinical applications. When designing a skin model there are various chemical and physical considerations that need to be considered to produce a biomimetic design. This includes designing a structure that mimics the structural characteristics and mechanical strength needed for tribological property measurement and toxicological testing. Presently, various commercial products have made significant progress towards achieving a native skin alternative, with focuses on one or more of the aforementioned considerations. Further research perspectives involve development of a functional bi‐layered model that mimics the constituent properties such as viscoelasticity and organization of the native epidermis and dermis, as well as incorporation of dynamic elements to mimic skin’s interactions with other organs. In this article, the skin models are divided into three categories: In Vitro Epidermal Skin Equivalents, In Vitro Full Thickness Skin Equivalents, and Clinical Skin Equivalents. A description of skin model characteristics, testing methods, applications, and potential improvements is presented. This article is protected by copyright. All rights reserved.
Article
Human skin pigmentation is a result of constitutive and facultative pigmentation. Facultative pigmentation is frequently stimulated by UV radiation, pharmacologic drugs, and hormones whereby leads to the development of abnormal skin hyperpigmentation. To date, many state‐of‐art depigmenting compounds have been studied using in vitro model to treat hyperpigmentation problems for cosmetic dermatological applications; little attention has been made to compare the effectiveness of these depigmenting compounds and their mode of actions. In this present article, new and recent depigmenting compounds, their melanogenic pathway targets, and modes of action are reviewed. This article compares the effectiveness of these new depigmenting compounds to modulate several melanogenesis‐regulatory enzymes and proteins such as tyrosinase (TYR), TYR‐related protein‐1 (TRP1), TYR‐related protein‐2 (TRP2), microphthalmia‐associated transcription factor (MITF), extracellular signal—regulated kinase (ERK) and N‐terminal kinases (JNK) and mitogen‐activated protein kinase p38 (p38 MAPK). Other evidences from in vitro assays such as inhibition on melanosomal transfer, proteasomes, nitric oxide, and inflammation‐induced melanogenesis are also highlighted. This article also reviews analytical techniques in different assays performed using in vitro model as well as their advantages and limitations. This article also provides an insight on recent finding and re‐examination of some protocols as well as their effectiveness and reliability in the evaluation of depigmenting compounds. Evidence and support from related patents are also incorporated in this present article to give an overview on current patented technology, latest trends, and intellectual values of some depigmenting compounds and protocols, which are rarely highlighted in the literatures.
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The effects of 4-hydroxyphenyl alpha-glucopyranoside (alpha-arbutin) and 4-hydroxyphenyl beta-glucopyranoside (arbutin) on the activity of tyrosinase from human malignant melanoma cells were examined. The inhibitory effect of alpha-arbutin on human tyrosinase was stronger than that of arbutin. The K(i) value for alpha-arbutin was calculated to be 1/20 that for arbutin. We then synthesized arbutin-alpha-glycosides by the transglycosylation reaction of cyclomaltodextrin glucanotransferase using arbutin and starch, respectively, as acceptor and donor molecules. The structural analyses using 13C- and 1H-NMR proved that the transglycosylated products were 4-hydroxyphenyl beta-maltoside (beta-Ab-alpha-G1) and 4-hydroxyphenyl beta-maltotrioside (beta-Ab-alpha-G2). These arbutin-alpha-glycosides exhibited competitive type inhibition on human tyrosinase, and their K(i) values were calculated to be 0.7 mM and 0.9 mM, respectively. These arbutin-alpha-glycosides possessed stronger inhibitory activity than arbutin, but less activity than alpha-arbutin. These results suggested that the alpha-glucosidic linkage of hydroquinone-glycosides plays an important role in the inhibitory effect on human tyrosinase.
Article
The effects of α- and β-arbutin on the activity of tyrosinases from mushroom and mouse melanoma were examined. α-Arbutin was synthesized from hydroquinone and starch using glucoside synthetase (GSase). β-Arbutin inhibited both tyrosinase activities from mushroom and mouse melanoma. α-Arbutin inhibited only the tyrosinase from mouse melanoma, 10 times as strongly as β-arbutin. The IC50 of α-arbutin was 0.48 mM and its inhibitory mechanism was speculated to be mixed type inhibition, while that of β-arbutin was noncompetitive.
Article
Transglycosylation from sucrose to phenolic and related compounds by sucrose phosphorylase (EC 2.4.1.7) from Leuconostoc mesenteroides was studied. The enzyme had a rather broad acceptor specificity and transferred glucosyl residue of sucrose to various acceptors such as hydroxybenzenes, hydroxybenzoic acids, benzyl alcohol, and hydroxybenzyl alcohols. The enzyme could transfer the glucosyl moiety of sucrose to phenolic OH groups and alcoholic (hydroxymethyl) OH groups, though not to carboxyl groups. The phenolic OH group was more effective than a hydroxymethyl group as the acceptor. Phenolic OH groups adjacent to hydroxyl, hydroxymethyl, or carboxyl groups had an increased effectiveness as acceptors.About 2.3 g of the purified transfer product was obtained from 2.0 g of hydroquinone. Its structure was identified as hydroquinone-O-α-d-glucopyranoside (α-arbutin) on the bases of the secondary ion mass spectrometry analysis, the component analysis of its enzymatic hydrolysates, and the carbon-13 nuclear magnetic resonance analysis. The browning resistance of α-arbutin to light irradiation was extremely increased compared to that of hydroquinone. The inhibitory activity on tyrosinase of α-arbutin was almost equal to that of arbutin.
Article
Melanoderm (Mat-Tek) is an in vitro model of the human epidermis consisting of well-differentiated, cultured human keratinocytes and melanocytes. We utilized this model to evaluate the efficacy, stability, and cytotoxicity of whitening agents. Magnesium ascorbyl phosphate (MAP), kojic acid, and lactic acid in aqueous or anhydrous base were applied to Melanoderm. Following incubation, tyrosinase activity was measured using L-dihydroxyphenylalanine (L-DOPA). Melanocyte staining was observed under the micro- scope. Melanoderm treated with either MAP, kojic acid, or lactic acid showed 33%, 48%, and 46% reduction, respectively, of tyrosinase activity. Microscopic examination of treated Melanoderm clearly showed the dendritic nature of melanocytes, and normal morphology of keratinocytes and MTT assay suggested that the test materials were not cytotoxic. The kojic acid effect declined with the age of the preparation, and subsequent analysis via high performance liquid chromatography (HPLC) showed kojic acid to be unstable in the aqueous base. Clinical tests using a chromameter to evaluate skin color indicated that kojic acid in an anhydrous base can induce more skin lightening than in the aqueous base. We obtained a good correlation between the Melanoderm, HPLC, and clinical tests. The data show that Melanoderm is a suitable tool for screening whitening agents and developing whitening products. A combination of two in vitro tests, such as the Melanoderm and HPLC methods, is useful to evaluate the relative activity, stability, and cytotoxicity of whitening ingredients and products before testing on humans.
Article
Six hundred strains of soil microorganisms were screened for the production of hydroquinone glucosylating enzyme (HGE). One of these strains, Bacillus subtilis strain X-23, produced an enzyme that glucosylated hydroquinone in the culture filtrate. The HGE was successively purified by ammonium sulfate fractionation, and Q-Sepharose, Phenyl-Toyopearl, and Superose 12 column chromatographies. The molecular weight was estimated as 65 kDa by SDS-polyacrylamide gel electrophoresis, and 54 kDa by gel filtration. Based on an analysis of the hydrolytic products from soluble starch, HGE was considered to be a kind of α-amylase. The structure of the hydroquinone glucoside produced by HGE was identified as by α- and β-glucosidase treatments, and 1H-NMR and 13C-NMR.
Article
We assessed the effects of arbutin on the pigmentation of cultured normal human melanocytes. As indicated by a cell-blotting assay, arbutin at concentrations in the range of 0.5-8 mM increased the pigmentation of the cultured melanocytes, while kojic acid at concentrations in the range of 0.5-4 mM decreased the pigmentation. The pigmentation-augmenting effect of arbutin was further confirmed by the results of a cell-pelleting assay, the traditional method of assessment. Treatment of the cells with arbutin increased the melanin content of the cells and the protein content as well. On the other hand, the tyrosinase activity in the cells was reduced by arbutin treatment. The levels of transcription of tyrosinase and tyrosinase related protein-1 genes were not affected by arbutin treatment as indicated by a semi-quantitative reverse transcription-polymerase chain reaction assay. These results demonstrate that arbutin promotes an increase in pigmentation of cultured human melanocytes that is not mediated by augmented tyrosinase activity.
Article
Four cases of primary malignant melanoma of the vagina in women aged 23, 44, 51 and 65 years are presented. In these 4 cases, thorough clinical and postmortem examinations ruled out the possibility of a primary melanoma elsewhere. The primary tumors showed exophytic growth with superficial ulceration. Three of the melanomas arose from the middle third of the vagina and one from the upper third. Melanin was visible in sections stained with hematoxylin and eosin in 3 of the tumors. In the other one, the first biopsy failed to reveal melanin. However, the second biopsy performed following irradiation showed abundant melanin pigment. Electron microscopic examination of 3 tumors revealed premelanosomes and melanosomes in the tumor cells, thus confirming the diagnosis. Two neoplasms showed atypical histologic features, and only the presence of melanin enabled us to make diagnosis of malignant melanoma. One melanoma was associated with an adjacent widespread intraepithelial component of superficial spreading type indicating its probable mode of origin. All 4 patients died of widespread metastases within 13 months after initial treatment. These 4 cases, in which clinical diagnosis was confirmed by thorough autopsy, strongly indicate that malignant melanoma can arise directly from the vagina.
Article
Normal human epidermal melanocytes became swollen and more dendritic with an increase in the amount of tyrosinase and immunoreactive b-locus protein when they were cultured for 2 days with the following arachidonic acid metabolites: prostaglandin (PG) D2, leukotriene (LT) B4, LTC4, LTD4, LTE4, thromboxane (TX) B2 and 12-hydroxy eicosatetraenoic acid (12-HETE). The effect of LTC4 was particularly strong compared to that of PGE2, about which we have previously reported. On the other hand, PGE1, PGF2 alpha and 6-ketoPGF1 alpha did not show any significant stimulatory effect. These data suggest that arachidonate-derived chemical mediators, especially LTC4, may be responsible for the induction of post-inflammatory hyperpigmentation of the skin.
Article
Melanin synthesis of B16 mouse melanoma cells was found to be stimulated dose and time dependently by 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], the hormonal form of vitamin D3. The stimulation of melanogenesis resulted from an increase in the activity of tyrosinase, a key enzyme in melanin synthesis. The minimum dose required for this stimulation was as low as 0.05 ng/ml, or 0.12 nM, a physiological level of plasma 1 alpha,25(OH)2D3. The stimulation by 1 alpha,25(OH)2D3 was specific; other derivatives of vitamin D3 caused no stimulation at a concentration of 500 ng/ml. When the cells were plated on agar plates, the proportion of dark or black colonies was not increased by the exposure to 1 alpha,25(OH)2D3. Furthermore, this compound did not induce melanin synthesis of an amelanotic variant. Thus, its stimulatory effect seemed to be due to stimulation of melanin synthesis of melanotic cells, rather than to conversion of amelanotic clones to melanotic ones. 1 alpha,25(OH)2D3 did not induce intracellular cyclic adenosine 3':5'-monophosphate, while cholera toxin induced cyclic adenosine 3':5'-monophosphate and stimulated melanin synthesis and tyrosinase activity much more than did 1 alpha,25(OH)2D3, suggesting that 1 alpha,25(OH)2D3 stimulates melanin synthesis by a cyclic adenosine 3':5'-monophosphate-independent mechanism. B16 melanoma cells contained specific receptors for 1 alpha,25(OH)2D3. Scatchard plot analysis revealed two types of receptor; the high-affinity receptor had a Kd of 18.3 pM and an Nmax of 10.6 fmol/mg of protein. The specificity of receptor binding was demonstrated by studies showing that, for 50% displacement of 1 alpha,alpha,25(OH)2D3 binding, other derivatives were required at 500 times higher concentrations or more. In contrast to 1 alpha,25(OH)2D3, retinoic acid inhibited melanin synthesis and tyrosinase activity of B16 melanoma cells dose and time dependently. On simultaneous treatment, 1 alpha,25(OH)2D3 and retinoic acid caused mutual interference, and a balance between their respective stimulating and inhibitory effects was obtained at a molar ratio of 10:1; i.e., with 10 nM 1 alpha,25(OH)2D3 and 1 nM retinoic acid.
Article
In previous studies we have shown melanotic melanomas to be exquisitely more sensitive to hydroquinone (HQ) inhibition than non-melanotic cell lines in vitro. Indeed, incorporation of [H3] Urd and [H3] Thd have been shown to be respectively 80 and 35 times more sensitive to HQ inhibition. The difference between the cell lines studied was their derivation, marked by their different melanin contents. The presence of melanin was proposed as a possible explanation of the differences. However, comparative experiments reported here demonstrate that amelanotic melanoma cell lines are equally susceptible to HQ inhibition. Thus, the action of HQ is apparently independent of the melanin content of the cell. Significantly, the tyrosinase levels in the melanomas and the amelanomas were found to be comparable and markedly different from that in the non-melanoma control cell lines. Thus, the results reported here support the hypothesis put forward by other workers that hydroquinone melanotoxicity is independent of cellular melanin content but requires the presence of active tyrosinase.
Article
Histochemical (dopa reaction) and electron microscopic studies were carried out to elucidate the nature of the chemical depigmentation produced by hydroquinone (HQ). Depigmentation was induced by topical application or subcutaneous injection of HQ in black guinea pigs. The present study showed that HQ preferentially affected the nonfollicular and follicular melanocyte system. It caused decreased formation of melanosomes, a marked alteration in the internal structure of melanosomes, an increased degradation of melanosomes , and , finally, a destruction of the membranous organelles in the melanocytes. These findings indicate that HQ affects not only the formation, melanization, and degradation of melanosomes, but that it affects also the membraneous structures of melanocytes and eventually causes necrosis of whole melanocytes.
Article
The effect of the skin-depigmenting agent hydroquinone (HQ) on 2 melanotic and 3 nonmelanotic cell lines was studied. Significant differences in its effect on DNA and RNA synthesis were observed between cell lines. HQ caused inhibition of cellular metabolism in all cells tested, but the dose that caused 50% inhibition of tritiated thymidine incorporation was approximately 30 times lower for melanotic cells. Tritiated uridine incorporation was found to be 85 times more sensitive to HQ in the melanotic cells. These results suggest that HQ exerts its depigmenting effect by selective action on melanocyte metabolism rather than a specific effect on melanin synthesis. Further, the effects of UV irradiation on this system were investigated and found to be negligible, in spite of the stimulation of in vivo melanin synthesis by UV radiation.
Article
In order to investigate the molecular basis of substrates, and the inhibitory specificity of tyrosinase, a large series of phenolic compounds have been analysed by using a High Performance Liquid Chromatographic-Scanning Spectrophotometric system. Depending on their chemical structure, phenolic compounds may act as substrates or as competitive inhibitors of tyrosinase. The ability to act as substrates requires the presence in the molecule of electron donor groups, while competitive inhibition on the contrary requires the presence of powerful electron acceptor groups. Certain phenolic compounds used as therapeutic agents or as food preservatives are chemically capable of acting as alternative substrates or competitive inhibitors of tyrosinase in vitro; their effect on melanocytes in vivo therefore merits investigation.
Article
Inhibitory effects of hydroquinone-alpha-glucoside (HQ-alpha-G) on the melanogenesis were investigated and compared with those of arbutin. The levels of inhibitory effects of HQ-alpha-G and arbutin on the tyrosinase activity were nearly the same. Inhibitory effects of both compounds on the melanogenesis, were studied using cultured B16 melanoma cells, and HQ-alpha-G was also found to have a similar effect to that of arbutin without inhibiting cell growth. In this experiment, while HQ-alpha-G hardly inhibited cell growth at 1 mM, arbutin inhibit it significantly at the same concentration. From these results it is suggested that HQ-alpha-G as well as arbutin inhibited the melanogenesis by affecting tyrosinase rather than by killing melanocytes. Furthermore, the melanogenesis of guinea-pigs with brown hair was reduced to about 80% by applying them each compound. The great differences in toxicity to normal human keratinocyte were not recognized between these two glucosides. It is, therefore, considered that HQ-alpha-G is an effective and safe ingredient for cosmetics.
Article
Arbutin, a naturally occurring beta-D-glucopyranoside of hydroquinone, is effective in the topical treatment of various cutaneous hyperpigmentations characterized by hyperactive melanocyte function. We examined the mechanism of its depigmenting action in human melanocyte cultures. Arbutin inhibited the tyrosinase activity of cultured human melanocytes at noncytotoxic concentrations. It did not affect the expression of tyrosinase mRNA. Melanin production was inhibited significantly by arbutin, as determined by measuring eumelanin radicals with an electron spin resonance spectrometer. The study of the kinetics and mechanism for inhibition of tyrosinase confirms the reversibility of arbutin as a competitive inhibitor of this enzyme. The utilization of L-tyrosine or L-dopa as the substrate suggests a mechanism involving competition with arbutin for the L-tyrosine binding site at the active site of tyrosinase. These results suggest that the depigmenting mechanism of arbutin in humans involves inhibition of melanosomal tyrosinase activity, rather than suppression of the expression and synthesis of tyrosinase.
Article
The inhibitory effect of arbutin, a naturally occurring beta-D-glucopyranoside derivative of hydroquinone, on melanogenesis was studied biochemically by using human melanocytes in culture. Cells were cultured in the presence of different concentrations of arbutin. The maximum concentration of arbutin that was not inhibitory to growth of the cells was 100 micrograms/ml. At that concentration, melanin synthesis was inhibited significantly by approximately 20% after 5 days, compared with untreated cells. This phenotypic change was associated with the inhibition of tyrosinase and DHICA polymerase activities, and the degree of inhibition was dose dependent. No significant difference in DOPAchrome tautomerase (DT) activity was observed before or after arbutin treatment. Western blotting experiments revealed there were no changes in protein content or in molecular size of tyrosinase, TRP-1 or TRP-2, indicating that inhibition of tyrosinase activity by arbutin might be due to effects at the post-translational level.
Article
Reconstituted 3-dimensional human skin equivalents containing melanocytes and keratinocytes on an artificial dermal substitute are gaining popularity for studies of skin metabolism because they exhibit morphological and growth characteristics similar to human epidermis. In this study, we show that such a pigmented epidermis model can be used to assess the regulation of pigmentation by known melanogenic compounds. In monolayers or in melanocyte-keratinocyte co-cultures, melanocyte-keratinocyte interactions are missing or are spatially limited. The commercial skin equivalents used in this study were derived from epidermal cells obtained from donors of three different ethnic origins (African- American, Asian, and Caucasian), and they reflect those distinct skin phenotypes. We used these pigmented human epidermis models to test compounds for potential effects on pigmentation in a more physiologically relevant context, which allows further characterization and validation of interesting melanogenic factors. We used known melanogenic stimulators (alpha-melanocyte-stimulating hormone and 3,4-dihydroxyphenylalanine) and inhibitors (hydroquinone, arbutin, kojic acid, and niacinamide) and examined their effects on the production of melanin and its distribution in upper layers of the skin. Our studies indicate that commercial skin equivalents provide a convenient and cost-effective alternative to animal testing for evaluating the regulation of mammalian pigmentation by melanogenic factors and for elucidating their mechanisms of action.
Article
Alpha-arbutin, a useful cosmetic ingredient, was selectively synthesized by alpha-anomer-selective glucosylation of hydroquinone with maltose as a glucosyl donor using lyophilized cells of Xanthomonas campestris WU-9701 as a biocatalyst. When 45 mM hydroquinone and 120 mg of lyophilized cells showing 11 nkat of alpha-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3NaOHKCl buffer (pH 7.5) containing 1.2 M maltose at 40 degrees C, only one form of hydroquinone glucoside was selectively obtained as a product and identified as hydroquinone 1-O-alpha-D-glucopyranoside (alpha-arbutin) by 13C-NMR, 1H-NMR and two-dimensional HMBC analysis. Although hydroquinone has two phenolic -OH groups at the para position in its structure, only one -OH group, but not both -OHs, was glucosylated and no other glucosylated products such as maltotriose were detected in the reaction mixture. The reaction at 40 degrees C for 36 h under optimum conditions yielded 42 mM alpha-arbutin, and the maximum molar conversion yield based on the amount of hydroquinone supplied reached 93%.
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