A Yeast Glycolipid Biosurfactant, Mannosylerythritol Lipid, Shows Potential Moisturizing Activity toward Cultured Human Skin Cells: The Recovery Effect of MEL-A on the SDS-damaged Human Skin Cells

A Yeast Glycolipid Biosurfactant, Mannosylerythritol Lipid, Shows Potential Moisturizing Activity toward Cultured Human Skin Cells: The Recovery Effect of MEL-A on the SDS-damaged Human Skin Cells

Abstract

Mannosylerythritol lipids (MELs) are produced in large amounts from renewable vegetable oils by Pseudozyma antarctica, and are the most promising biosurfactants known due to its versatile interfacial and biochemical actions. In order to broaden the application in cosmetics and pharmaceuticals, the skin care property of MEL-A, the major component of MELs, was investigated using a three-dimensional cultured human skin model. The skin cells were cultured and treated with sodium dodecyl sulfate (SDS) solution of 1 wt%, and the effects of different lipids on the SDS-damaged cells were then evaluated on the basis of the cell viability. The viability of the damaged cells was markedly recovered by the addition of MEL-A in a dose-dependent manner. Compared to the control, MEL-A solutions of 5 wt% and 10 wt%gave the recovery rate of 73% and 91%, respectively, while ceramide solution of 1 wt% gave the rate of over 100%. This revealed that MEL-A shows a ceramide-like moisturizing activity toward the skin cells. Considering the drawbacks of natural ceramides, namely limited amount and high production cost, the yeast biosurfactants should have a great potential as a novel moisturizer for treating the damaged skin.

INTRODUCTION

The skin roughening, namely dry skin is generally caused by the damage to the intracellular lipids of the skin, which induces the loss of water–retention properties of the stratum corneum. This kind of damage is often triggered by improper use of detergents and/or cleaners. For example, sodium docecyl sulfate (SDS) were reported to induce the disorganization of the stratum corneum through the marked increase of transdermal water loss1). Such surfac-tant–induced dry skin also shows the loss of the intracellular lipids accompanied by the disruption of multiple lamella structure.

Intracellular lipids such as sphingolipids are considered to play an essential role in the maintenance of water–reten-tion capacity in the stratum corneum2). Ceramides, the key intermediates in the biosynthesis of sphingolipids, have been received increasing attention as an effective moisturizing ingredient for cosmetics and pharmaceuticals, because of the potential water–retention properties. How-ever, the quantity of natural ceramides in organisms is very limited, and thus the large–scale preparation of the lipids from natural resources seems considerably difficult.

Synthetic ceramides are obtained by acylation of the amine group of sphinganine or its derivative, and widely used as moisturizers alternative to natural ones. However, these synthetic approaches for the large–scale preparation of isomerically pure ceramides are tedious and time consuming for commercial applications3). It is thus of great interest to develop a novel and cost–effective natural moisturizer alternative to ceramides.

Mannosylerythritol lipids (MELs, Fig. 1) are promising glycolipid biosurfactants, and are abundantly produced at a yield over 100 g/L from vegetable oils by different yeast strains of the genus Pseudozyma4,5). MELs efficiently form different lyotropic liquid crystalline phases including a lamellar (La) in a broad range of concentration6–8). In addition, MEL–A, the major component of MELs, has the similar amphiphilic structure to that of ceramide–3 (Fig. 1) and shows the similar biochemical actions to those of glycosph-ingolipids4). These facts imply that MEL–A would exhibit ceramide–like skin care properties. We thus focused our attention on the feasible use of MEL–A as a moisturizer instead of ceramides and their derivatives.

Novel test methods using no animals have been desired for investigating the skin care properties of cosmetic mate-rials. Recently, living skin equivalent, which consists of three layers of human skin cells (the stratum corneum, epi-dermal, and dermal cells) in a three–dimensional matrix, has been developed as a practical human skin model9,10). In this study, we thus employed a commercially available three–dimensional cultured human skin model for evaluating the moisturizing activity of MEL–A.

Here, we report for the first time the recovery effect of MEL–A on the SDS–damaged human skin cells, and demonstrated that the present yeast biosurfactant has a great potential as a novel and cost–effective moisturizer.

Fig. 1

Chemical Structure of Mannosylerythritol Lipids (MELs) and Ceramide3.

EXPERIMENTAL

2.1 Production of glycolipid, MEL-A

Pseudozyma antarctica T–34 was cultured in a growth medium [4% glucose, 0.3% NaNO3 , 0.03% MgSO4, 0.03%KH2PO4, 0.1% yeast extract (pH 6.0)] at 25˚C on a reciprocal shaker (150 strokes/min) for 2 d11). The obtained seed culture (0.1 mL) was transferred to a 200–mL Erlenmeyer flask containing 20 mL of a production medium [5% soy-bean oil, 0.3% NaNO3, 0.03% MgSO4 , 0.03% KH2PO4, 0.1%yeast extract (pH 6.0)] at 25˚C on a rotary shaker (250 rpm) for 7 d. The produced glycolipids were extracted from the culture medium with an equal amount of ethyl acetate.

2.2 Purification of MEL-A

The above organic layer was separated and evaporated. The concentrated MELs were dissolved in chloroform and then purified by silica–gel (Wako–gel C–200) column chromatography using a gradient elution of chloroform/acetone (10:0 to 0:10, vol/vol) mixtures as solvent systems12). The purified MEL–A was used in the following experiments.

2.3 Quantification of MEL-A by high-performance liquid chromatography (HPLC)

The quantification of MEL–A was carried out by HPLC on a silica gel column (Inertsil SIL 100A 5 µm, 4.6 ( 250 mm; GL science Inc, Japan) with a low temperature–evaporative light scattering detector (ELSD–LT; Shimadzu, Japan) using a gradient solvent program consisting of various pro-portions of chloroform and methanol (from 100: 0 to 0: 100, vol/vol) at a flow rate of 1 mL/min13). The quantification of MEL–A was based on the standard curve using the pure MEL–A fraction as described previously12).

2.4 Structural analysis

The structure of the purified MEL–A was confirmed by 1H nuclear magnetic resonance (NMR) with a Varian INOVA 400 (400 MHz) at 30˚C using the CD3OD solution. The fatty acid profile of the MEL–A was analyzed by gas chromatography–mass spectrometry (GC–MS) (Hewlett Packard 6890 and 5973N) with a TC–WAX (GL–science, Japan) with the temperature programmed from 90˚C (held for 3 min) to 240˚C at 5˚C/min, as described previously14).

2.5 Viability assay

The moisturizing activity of MEL–A was evaluated using a three–dimensional cultured human skin model, TEST-SKINTM (Toyobo, Japan), on the basis of the cell viability. SDS solution (1 wt%) was applied to the cell surface and incubated for 5 min at 37˚C, in order to induce the dry skin conditions. After washing out the SDS solution with fresh culture medium (1 mL), olive oil fraction (50 µL) containing various concentrations of MEL–A was directly applied, and then the cells were incubated for 24 h at 37˚C. Ceramide from bovine brain (Sigma, Japan) was purchased from Wako (Japan) and used as the positive control. The viability of cells was colorimetrically determined using a MTT assay kit (Nakalai, Japan). The cells cultured without the SDS treatment was used as the positive control.

Fig. 2

Protection of the Cultured Skin Cells from SDS-induced Damage by MEL-A. The cultured skin cells were treated with 1% SDS, washed out the SDS solution, and then re-treated with MEL-A. The living cells were detected by a colorimetric method, MTT assay.

Fig. 3

Relative Viability of the Cultured Skin Cells Treated with SDS. The cultured skin cells were treated with 1% SDS, washed out the SDS solution, and then re-treated with MEL-A dissolved in olive oil. The viability of cells was determined with a colorimetric method (MTT assay) at 570 nm. Ceramide was used as the positive control. -SDS: non-treated with SDS, +SDS: treated with SDS.

RESULTS AND DISCUSSION

MELs were produced from soybean oil as the sole carbon source by P. antarctica T–34. The purified MEL–A was obtained by the silica–gel column chromatography and subjected to NMR analysis. The structure of MEL–A was con-firmed as “di–acetylated and di–acylated 4–O–β–D–mannopy-ranosyl–meso–erythritol”. Based on GC–MS analysis, the main fatty acids of MEL–A were C8 (26%) and C10 (73%), which corresponded well with the data on the previous reports5,14). The purified MEL–A was thus exclusively used in the following experiments.

In order to estimate the skin care property of MEL–A, MEL–A was subjected to the cultured human skin model, and the cell viability was determined by the MTT method (Fig. 2). The control cells cultured without the SDS treatment showed a blue color reaction indicating a good viability, whereas the SDS–treated cells hardly showed the color reaction. This means that the SDS treatment clearly lower the cell viability and induce the dry skin condition.

SDS treatment showed the blue color reaction (Fig. 2). The recover y effect of MEL–A on the cells from the SDS–induced damage was observed in a dose–dependent manner. Compared to the control, the cell viability treated with MEL–A solutions of 0.1, 1, 5, 10 wt% were 18.4, 36.2, 73.2, 91.3%, respectively (Fig. 3). Olive oil alone gave little effect on the cell viability after the SDS treatment. In the case of ceramides, the cells treated with 0.1 wt% solution showed little effect on the viability, while the cells with 1 wt% solution completely recovered from the SDS–induced damage. These results suggested that MEL–A has ceramide–like skin care property. The recovery effect of MEL–A toward the SDS–damaged cells is probably due to the moisturizing activity, considering the well–known skin care properties of ceramides. Skin ceramide play an important role in the water permeability properties of the skin, providing an epidermal water barrier which strengthens the skin structure and reduces water loss1-3).

Regarding the mechanism for the recovery effect, we also speculate 1) the structure of MEL–A resembling ceramides would allow the skin cells to be penetrated easily into the intercellular spaces, and 2) the penetrated MEL–A would effectively provide the moisture retention in the cells due to the excellent formation of liquid crystals7).

In conclusion, MEL–A would have a great potential for a novel skin care material, which possesses not only a potential moisturizing activity but also an advantage in the largescale preparation.

ACKNOWLEDGMENTS

The authors are also grateful to Ms S. Ishimaru and Mr. A. Nakagawa in Corporate Research Center of Toyobo Co., Ltd. for their help in the development of SDS–damaged human skin model.

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