Wang Zheng,Liu Jianning,Guo Yiguang,Qiao Xiaoling

Shanghai Jahwa United Co.Ltd.,China

Raoul Vyumvuhore,Laurie Verzeaux,Brigitte Closs

SILAB,R＆D Department,Brive la Gaillarde,France

Abstract

Skin moisturization can be measured in vivo by Corneometer®,giving information about the hydration level of the skin.However,this approach cannot reveal the inner changes of lipid organization involved in the integrity of the skin barrier.Raman microspectroscopy is an in vivo and non-invasive method that assesses the lateral organization of lipids from the stratum corneum by measuring the νasymCH2/νsymCH2 ratio.

In the present study,the aim is to evaluate the moisturizing effect of two skin care products (i.e.LOTION 1: GF deep-nourishing ＆ skin-sturdy facial emulsion;LOTION 2: placebo formula) by using the Corneometer® and Raman microspectroscopy.Results demonstrates a significant improvement of lipids organization and thus a reinforcement of the barrier function associated with an increase of the hydration level with LOTION 1 compared to LOTION 2.

Key words

skin barrier;Raman microspectroscopy;lipid organization;moisturization

Introduction

Skin is the barrier coating on the surface of the body,which maintains the balance of human body.Skin acts as an effective barrier against unwanted environmental influences,meanwhile,it prevents excessive water and electrolyte loss from the body.[1]The water content in normal skin ranges from 20 to 35%,but skin will become dry,rough and flaked off if the water content is below 10%.An effective skin barrier is very important for the healthy skin and it keeps skin away from disease.[2]The specific composition and structure of skin plays an important role in the barrier function.The epidermis is the outermost layer of the skin which consists of four distinctive layers in most parts of human skin.Each layer displays one of the sequential differentiation stages of the keratinocytes,the major cell type in the epidermis.The layers include thestratum corneum(SC),stratum granulosum(SG),stratum spinosum(SS),and the inner moststratum basale(SB).The upper layer of the skin is termed as SC,a layer with a 10-15μm thickness,responsible for the primary barrier of the skin.Human SC contains 10 to 25 corneocyte layers that are oriented approximately parallel to the skin surface and are embedded in a lipid matrix.The structure of the SC is often referred as a “bricks in mortar” structure,where corneocytes are the bricks and the lipids are the mortar.[3-6]The lipid regions are the only continuous structure in the SC,and considered to be very important for the skin barrier function.[7]The impermeable character of the hydrophilic cornified envelope in concert with the chemically linked lipophilic monolayer directs the penetration of most substances along the tortuous pathway between the corneocytes as revealed by confocal laser scanning microscopy and X-ray microanalysis studiesin vitroas well asin vivo.[8,9]Due to this,the intercellular lipid regions play a dominant role in the skin barrier and the arrangement of lipid regions dominates a key process in the formation of the skin barrier.

The development of modern non-invasive skin measuring techniques provides several objective and/or quantitative methods to investigate the skin barrier function.Transepidermal Water Loss (TEWL) is one of the most commonly used method.It measures the loss of water that passes from the body through the epidermis to the surrounding atmosphere via diffusion and evaporation processes.Thus,the increase of the TEWL value can indicate the damage of skin barrier function.Another tool frequently used to evaluate the skin hydration is the Corneometer®,allowing to determine the level of skin moisturization by the measurement of capacitance.[10]However,these methods cannot reveal the inner changes of skin structure and its functions.[11]But the Raman microspectroscopy can give information about the lipids organization of the skin and its barrier function changes.Recently,Raman microspectroscopy has been used to investigate the lipids organization in normal and dry skins,which demonstrates an impairment of the packing of lamellar lipid structures with the dryness.[12]The measurements focus on the main marker of lipids molecular organization,the νasymCH2/νsymCH2ratio.[13-15]This ratio is high in a compact lipid matrix of the SC (organized lipids) and its use for lipid study investigation has been confirmed by correlation of vibrational spectroscopic methods (Raman,Infrared) with other independent measurements like X-ray diffraction or differential scanning calorimetry.[15,16]

In this paper,we evaluated the moisturizing effects of two skin care products (LOTION 1: GF deep-nourishing ＆ skin-sturdy facial emulsion and LOTION 2: placebo formula) by Raman microspectroscopy and Corneometer®by applying two times a day for 4 weeks.

Materials and methods

Anin vivostudy using Raman microspectroscopy and Corneometer®was conducted to determine the change of skin barrier function after using a market product,LOTION 1 (i.e.GF deep-nourishing ＆ skin-sturdy facial emulsion) or LOTION 2 (i.e.Placebo formula,which is LOTION 1 without adding OPHIOPOGON JAPONICUS ROOT EXTRACT).

Testing protocol

The study included 24 healthy male volunteers between 25 and 64 years of age,selected with dry skin (mean hydration rate of the LOTION 1 group at D0: 25.3,mean hydration rate of the LOTION 2 group: 27.9,according to a measurement using the Corneometer®CM825 (Courage+Khazaka electronic GmbH)) on the external-anterior-lateral side of the arm.

The volunteers applied testing products on their external-anterior-lateral side of the arm twice a day (morning and evening) during 4 weeks by applying in light massage until the penetration according to the information indicated on the bottle.The measurements were performed for 15 minutes of acclimation under control room conditions (22°C+/-2°C and 40 to 60% of relative humidity).

Raman microspectroscopy experimental system Acquisition of spectra

The system was composed of a Raman confocal probe (Horiba Jobin-Yvon) coupled with a dispersive Raman spectrometer (Micro HR,Horiba Jobin-Yvon).The excitation laser beam was sent to the measurement probe through a 5 μm diameter monomode fiber and the Raman signal was sent to the spectrometer through a 100 μm diameter fiber.The probe was equipped with a 100X long focal working objective (MPlan FLN,Olympus) operated in air with a numerical aperture of 0.9.A piezoelectric system (Physics Instrument) collected the Raman profiles in the Z axis,from the surface of the skin down to a predefined depth.Axial resolution of the system was about 3 μm (manufacturer's specification).A video camera in the probe was used to visualize the surface of the skin and also to control laser beam focusing on the surface of the skin.The spectrometer was equipped with a CCD camera (Charge Coupled Detector) (Synapse,Horiba Jobin-Yvon) with 1024 X 256 resolution,chilled with the Peltier effect,and with a grating of 830 lines/mm covering a broad spectral range of 400 to 3,620 cm-1in a single acquisition with a spectral resolution of about 7 cm-1.The excitation source was a laser diode at 660 nm (Ignis Laser Quantum GmbH).Output power of the objective (at the level of the sample) was set at 20 mW,consistent with standards,governing protection from radiation.The excitation wavelength of 660 nm was selected because it was an optimal compromise between the generation of parasite fluorescence and sensitivity of the CCD camera over the entire spectral range of interest.The acquisition system was managed by Labspec 6 software (Horiba Jobin-Yvon).The principal difficulties of allin vivomeasurements involved the movements of the body that can affect laser focusing.In order to optimize measurement conditions,the extremity of the probe was equipped with a system independent of the objective for maintaining its position that was in contact with the surface of the skin.A 1 mm diameter hole was drilled in the head of the probe to obtain acquisitions without having to use glass slides on the surface of the skin.Raman profiles were recorded by collecting spectra starting from 10 μm above the surface of the skin and down to the depth of 30 μm.

The localization of sites and their identification at kinetic time points were ensured by the use of a locator mask (Transparent positioning sheet,Monaderm).Measurements were conducted on symmetrical zones of the external-anterior-lateral side of the arm.

Data analysis

Spectral data were pre-processed with in house software operating in Matlab®environment (Matlab®7.2 software (The MathWorks).These algorithms were developed by the team of Prof.Michel Manfait (MeDIAN-CNRS UMR7369,Reims Champagne-Ardenne School of Pharmacy).Aberrant profiles were excluded by visual inspection of the data.Selected profiles underwent a series of corrections to “clean up” the Raman signal from the skin.This step included a correction of the signal from the optical system and a correction of the baseline using a fourth order polynomial function.Raman profiles then underwent a correction of axial position because of a slight laser unfocusing that may occur between adjustment of the probe on the surface of the skin and the start of measurements.The position of the surface of the SC in this step was calculated from Raman acquisitions by determining the Z position corresponding to the half-maximum intensity of CH vibrations (intensity integrated between 2,800 and 3,000 cm-1).Finally,Raman spectra were smoothed with a Savitzky-Golay filter (second order polynomial function in a window of 9 wavenumbers) and vectorially normalized over the entire spectral range.The vasymCH2/vsymCH2ratio was then studied.The rand features test was used to show discriminating frequencies.

Study of the moisturizing effect

Measurement by Corneometer®

Skin hydration levels of the external-anteriorlateral side of the arm were measured with the Corneometer®CM825.The probe of this device quantitatively and directly measured the electric capacitance of the skin,in other words,the capacity of intercellular water in the stratum corneum to conduct electrons.This parameter was directly linked to the hydration status of the skin.The localization of sites and their identification at kinetic time points was ensured by the use of a locator mask (Transparent positioning sheet,Monaderm).Measurements were conducted on symmetrical zones of the externalanterior-lateral side of the arm.

Data processing

The effect of the LOTION 1: GF deep-nourishing ＆ skin-sturdy facial emulsion formula and LOTION 2: placebo formula was determined by examining changes between D0 and D28,i.e.the variation between before and after treatment.

With: MV (D0): mean value on the zone treated with the LOTION 1 and LOTION 2 before application.MV (D28): mean value on the zone treated with the LOTION 1 and LOTION 2 after 28 days of twicedaily applications.

The effect of the LOTION 1 compared to the LOTION 2 was determined by the difference between the change with product and the placebo:

With: (Δ / D0) Product: change compared to D0 on the zone treated with the LOTION 1 after 28 days of twice-daily applications.(Δ / D0) Placebo: change compared to D0 on the zone treated with the LOTION 2 after 28 days of twice-daily applications.

Results and discussion

Study of barrier function by Raman microspectroscopy

Comparative study of mean spectra on area treated with LOTION 1: GF deep-nourishing ＆ skin-sturdy facial emulsion formula at D0 and D28

Figure 1 showed the mean Raman microspectroscopy spectra of the external-anterior-lateral side of the arm before (D0) and after 28 days of treatment (D28) with the LOTION 1.Regions shown by a blue line are spectral bands containing differences between the two mean spectra.

Figure 1.Mean spectra of the LOTION 1: GF deepnourishing ＆ skin-sturdy facial emulsion formula areas before and after 28 days of twice-daily applications

In the spectral range of the νasymCH2and νsymCH2vibrations,we observed the significant differences between D0 and D28 on areas treated with the LOTION 1.This result demonstrated that the LOTION 1 affects the lipids organization after 28 days of use.

Comparative study of mean spectra on zone treatedwith the LOTION 2: placebo formula at D0 and D28

Figure 2 showed the mean Raman microspectroscopy spectra of the external-anterior-lateral side of the arm before (D0) and after 28 days of treatment (D28) with the LOTION 2.Regions shown by a blue line correspond to spectral bands containing differences between the two mean spectra.

Figure 2.Mean spectra of placebo areas before and after 28 days of twice-daily applications

In the spectral range related to the lipids organization (νasymCH2and νsymCH2vibrations),there was no statistically significant differences between D0 and D28 on areas treated with placebo.This result indicates that the placebo formula has no effect on lipids organization.

Comparative analysis between LOTION 1 and LOTION 2

Figure 3 summarized the results on lipids organization obtained after quantitative analysis of the Raman spectra.Results highlighted a significant variation of the lipids organization after 28 days of twice-daily applications,the LOTION 1 in comparison with the LOTION 2.

Figure 3.Effect of the LOTION 1: GF deep-nourishing ＆ skin-sturdy facial emulsion formula and the LOTION 2: placebo formula on lipids organization after 28 days of twice-daily applications

Study of the hydration

The hydration level of the skin was evaluated with a Corneometer®after 28 days of use of the LOTION 1 in comparison with the application of the LOTION 2.Figure 4 summarized the results of this study and demonstrated a significant increase of the skin hydration after 28 days of use of the LOTION 1,comparatively to the placebo formula.

Figure 4.Effect of the LOTION 1: GF deep-nourishing ＆ skin-sturdy facial emulsion formula and the LOTION 2: placebo formula on hydration of the skin after 28 days of twice-daily applications

Conclusion

The aim of this study was to demonstrate the efficacy of the LOTION 1: GF deep-nourishing ＆ skinsturdy facial emulsion formula comparatively to the LOTION 2: placebo formula on the skin moisturization.For this purpose,the barrier function was investigated by analyzing the lipids organization in the stratum corneum with an innovative approach using Raman microspectroscopy.Moreover,an evaluation of the skin hydration was assessed with a Corneometer®.Results highlighted that LOTION 2 improved the skin hydration.However,LOTION 2 could not increase the lipids organization,while LOTION 1 significantly improved it comparatively to the LOTION 2.Indeed,results indicated that after 28 days of twice-daily applications of the LOTION 1,there was an increase of the νasymCH2/νsymCH2vibrations ratio,revealing that the molecules were in close interaction with each other,reflecting a compact lipids organization and thus a more effective barrier function.The skin hydration measured by the Corneometer®was also significantly increased after 28 days of a twicedaily application of the LOTION 1.Altogether,these results demonstrated a moisturizing effect of the LOTION 1 compared to the LOTION 2,linked to an improvement of the lipids organization with the LOTION 1.The better efficacy of this lotion could be explained by the ingredients of the formulation,which contains glycerin,OPHIOPOGON JAPONICUS ROOT EXTRACT,betain and allantoin.

Acknowledgements

We would like to thank SILAB for the excellent collaboration and experiments execution.