Ceramidas
Ceramidas
Ceramidas
XXX
Blackwell
Oxford,
International
IJD
©
1365-4632
0011-9059
2008 The
UK
Publishing
International
Journal Ltd
of Dermatology
Society of Dermatology
International Journal of Dermatology 2008, 47, 812–819 © 2008 The International Society of Dermatology
Huang and Chang Ceramides and skin barrier function Report 813
lipid-rich emollients containing ceramide 3 may be of benefit glucose, sodium lauryl sulfate (SLS), and sodium lauroyl lactylate
in skin barrier disruption following tape stripping.16 Cera- were obtained from Sigma-Aldrich, Deisenhofen, Germany;
mides are extremely water-insoluble compounds, which can cyclomethicone, ceramide 1, ceramide 3, and carbomer were
form a water-impermeable barrier in the skin. Therefore, it purchased from Elgin Co., Ltd., Taipei, Taiwan; petrolatum, sucrose
is possible for ceramides to replace the depleted stratum stearate, sucrose distearate, dimethiconol, tromethamine, sodium
corneum lipids via a topical route. The problem with the hydroxymethylglycinate, and xanthan gum were provided by LICA
topical application of skin care products, however, is how to Enterprises Corp. Co., Ltd., Taipei, Taiwan; hydrogenated vegetable
deliver the active ingredients, such as ceramides, in sufficient oil was supplied by ESSENCE PLUS Co., Ltd., Hsin-Chu, Taiwan.
amounts to the target site.17
The aim of this study was to evaluate the effect of cera- Production of ceramide-containing emulsions and control
mides 1 and 3 on skin properties, such as skin hydration and emulsion
TEWL, in healthy Asian women. In addition, the relationship Stable (for 15 months) oil-in-water (o/w) emulsions incorporating
between the types of ceramide in cosmetic formulations and ceramide and phytosphingosine or phytosphingosine alone were
the barrier function of the stratum corneum was examined. successfully developed in our laboratory and designated as
emulsion A, emulsion B, emulsion C, and control emulsion.
Emulsion A contained ceramide 1, emulsion B contained ceramide
Materials and Methods
3, and emulsion C contained both ceramides 1 and 3. The control
Materials emulsion was produced by the same process, but ceramides 1
Sorbitol, glycerine, phenoxyethanol, citric acid, benzyl alcohol, and 3 were not included (Table 1). The preparation of the above
sodium hydroxide, sorbic acid, stearyl alcohol, caprylic/capric emulsions was performed as follows. The aqueous phase
triglyceride, dextrin, squalane, phytosphingosine, cholesterol, [containing glycerine (5.0%), sodium hydroxymethylglycinate
Table 1 Composition of the emulsions in this study. Emulsion A, ceramide 1-containing emulsion; emulsion B, ceramide 3-containing
emulsion; emulsion C, ceramide (1 + 3)-containing emulsion
Oil phase
Petrolatum 3.0 3.0 3.0 3.0
Hydrogenated vegetable oil 5.0 5.0 5.0 5.0
Cyclomethicone 3.0 3.0 3.0 3.0
Caprylic/capric triglyceride 3.0 3.0 3.0 3.0
Sucrose distearate 2.0 2.0 2.0 2.0
Dextrin 0.5 0.5 0.5 0.5
Squalane 2.0 2.0 2.0 2.0
Sucrose stearate 3.0 3.0 3.0 3.0
Candelilla cera 1.0 1.0 1.0 1.0
Dimethiconol 0.2 0.2 0.2 0.2
Phytosphingosine 0.005 0.005 0.005 0.005
Cholesterol 0.02 0.02 0.02 0.02
Ceramide 1 0.02 – 0.02 –
Ceramide 3 – 0.02 0.02 –
Water phase
Glycerine 5.0 5.0 5.0 5.0
Sodium hydroxymethylglycinate 0.1 0.1 0.1 0.1
Citric acid 0.1 0.1 0.1 0.1
Glucose 0.2 0.2 0.2 0.2
Sorbitol 3.0 3.0 3.0 3.0
Sodium lauroyl lactylate 3.0 3.0 3.0 3.0
Neutralizing agent
Tromethamine 0.3 0.3 0.3 0.3
Antiseptics
Benzyl alcohol 0.1 0.1 0.1 0.1
Phenoxyethanol 0.1 0.1 0.1 0.1
Viscosity enhancing agents
Carbomer 0.45 0.45 0.45 0.45
Xanthan gum 0.4 0.4 0.4 0.4
Distilled H2O 63.005 63.005 62.985 63.025
© 2008 The International Society of Dermatology International Journal of Dermatology 2008, 47, 812–819
814 Report Ceramides and skin barrier function Huang and Chang
(0.1%), citric acid (0.1%), glucose (0.2%), sorbitol (3.0%), sodium barrier function of the skin by removing and rearranging stratum
lauroyl lactylate (3.0%), xanthan gum (0.4%), carbomer (0.45%), corneum lipids. It has been suggested that the quantity and
and distilled water] was heated to 80 °C with slight mixing. spectrum of lipids removed from the stratum corneum are
Petrolatum (3.0%), hydrogenated vegetable oil (5.0%), detergent specific.19
cyclomethicone (3.0%), caprylic/capric triglyceride (3.0%), sucrose The skin studies were designed as one-sided, blind,
distearate (2.0%), dextrin (0.5%), squalane (2.0%), sucrose placebo-controlled studies with intraindividual comparison of
stearate (3.0%), dimethiconol (0.2%), phytosphingosine two formulations and two untreated test areas on the forearms. For
(0.005%), cholesterol (0.02%), and ceramide [ceramide 1 (0.2%), each study, the same 15 healthy Asian women (age, 20–30 years)
ceramide 3 (0.2%), or both] were mixed and heated until complete with healthy skin applied the two test emulsions twice daily over a
dissolution in the oil phase. The two phases were merged and period of 28 days. Each volunteer applied 0.2 g of the emulsions
homogenized with a high-pressure vacuum homogenizer with and without ceramide on each forearm. The skin properties
(General Purpose Pressure Vessels, Parr Instrument Company, were measured at approximately the same time on days 0
lilinois, USA) at 80 °C, 3000 r.p.m. for 15 min, and then cooled (baseline value, prior to the application of the emulsions), 1, 2, 3,
to 70 °C. Tromethamine (0.3%) was dissolved in water and added 7, 14, 21, 28, 29 (first day after last application), and 30 (second
to the 70 °C mixture described above, and homogenized at day after last application).
3000 r.p.m. for 10 min. The homogeneous emulsion was then
cooled to 45 °C. At the adjusted temperature, antiseptics, such as Evaluation
phenoxyethanol (0.1%) and benzyl alcohol (0.1%), were added To provide meaningful statements on the effects of the emulsions,
and further homogenized at 3000 r.p.m. for 10 min. After cooling to the results were calculated using the following equation
35 °C, the emulsion was stored at 25 °C. All emulsions were tested
Change in skin properties (%) = [(Pt – Po)/Po] × 100
further for their stability.
where Pt is the mean of the quotients of the measured values
Stability test of treated and untreated skin after application time t in all
The emulsions prepared in the study were examined using volunteers, and Po is the mean of the quotients of the measured
cosmetic stability tests, such as high temperature, low values of treated and untreated skin before application in all
temperature, room temperature, and circulating temperature. volunteers. The volunteers recorded the application time and
The stability tests should be maintained for at least 15 months. weight of the samples before and after the study at home. During
First, the emulsion was stored at 50 °C for 1 month. Second, the measurements, the volunteers were accommodated in an
the product was stored at 45 °C for 3 months. Third, the emulsion air-conditioned room at 20 ± 1 °C and 50 ± 5% relative humidity.
was stored at 25 °C for 1 year. Finally, the product was stored at
10 °C for 1 month. Skin hydration
The measurement of skin humidity was performed with a
Skin irritation and skin erythema Corneometer®825, which was mounted on a Multi Probe
Skin irritation was tested by patch test and observed visually. Adapter® MPA5 (Courage and Khazaka Electronic GmbH).20
Any type of change of the skin surface was recorded. The The measurement of skin hydration was carried out by the
ceramide-containing emulsions and control emulsion were capacitance method, which utilizes the relatively high dielectric
tested; SLS was used as a positive irritant control. Erythema constant of water compared with that of other substances in the
of the skin was measured using a Mexameter®18 (Courage skin. The measuring condenser was on the front surface of the
and Khazaka Electronic GmbH, Cologne, Germany), which measurement sensor. When the measurement head was pressed
determines the hemoglobin content of the skin photometrically onto the skin for 1 s, the horny layer came into the scatter range
based on the emission principle. The probe of the Mexameter®18 of the condenser field. The measurement of capacitance is
emits light of two defined wavelengths: 568 nm, which responds to entirely dependent on the water content in the skin. Different
the particular spectral absorption peak of hemoglobin; 660 nm, capacitance changes are converted into a digital measured value
which avoids other color influences. Light reflected by the skin (arbitrary units) which is proportional to the skin humidity. As the
was determined using a receiver. measurement time is short, measurement errors caused by skin
deformations or evaporation build-up can be excluded. For each
SLS irritation and application of the emulsions tested area, five measurements were performed at different points
In this study, all volunteers were exposed to 17% SLS for 7 h to on the forearm.
induce skin irritation, as described previously,18 and the emulsions
were then applied to the forearm. On removal of the SLS patches, TEWL measurements
the skin was gently rinsed with water and allowed to dry. The To measure the TEWL value of the skin, a TEWAMETER TM210
emulsions were then applied to the SLS-treated areas twice daily (Courage and Khazaka Electronic GmbH, Cologne, Germany)
and the skin properties were measured. SLS may disrupt the was used, according to the guidelines provided by the
International Journal of Dermatology 2008, 47, 812–819 © 2008 The International Society of Dermatology
Huang and Chang Ceramides and skin barrier function Report 815
Statistical analysis
Statistical analysis of the experimental data points was performed
by Wilcoxon’s signed rank test, which was used for comparison of
measured data employing SAS 9.0 statistical software. Differences
were considered to be statistically significant at P < 0.05.
Results
Skin hydration determination Figure 2 Effects of emulsion B () and control emulsion () on
In the skin hydration study, the results obtained with skin hydration. All results are expressed as values relative to the
ceramide-containing and control emulsions were compared. baseline measurement on day 0 and the untreated control
(mean ± standard deviation; n = 15). Significant differences
After treatment with emulsion A and control emulsion, skin
between the results: *P < 0.0001
hydration (%) increased in a time-dependent manner, with a
maximum increase in skin humidity obtained on day 28: 10.4
± 1.0% and 9.98 ± 1.1% for emulsion A and control emulsion,
respectively (Fig. 1). Two days after the last application, the
in increasing the water content of the skin. Figure 2 shows a
increases in skin humidity were 8.8 ± 0.8% and 8.4 ± 0.9%
similar effect of emulsion B and control emulsion on skin
for the same emulsions (Fig. 1), indicating the sustained effect
hydration, the maximum increase in skin humidity being
of the two emulsions on skin hydration. Skin hydration
reached after 4 weeks: 9.6 ± 0.6% and 8.8 ± 0.8% for emulsion
improvement by emulsion A was only slightly more effective
B and control emulsion, respectively (Fig. 2). Furthermore,
than that of control emulsion, indicating that ceramide 1 con-
the increase in skin humidity produced by both emulsions had
tained in emulsion A does not seem to play an important role
a sustained effect on skin hydration. No apparent difference
was observed between emulsion B and control emulsion.
Therefore, ceramide 3 in emulsion B does not seem to play an
important role in skin hydration.
For emulsion C and control emulsion, the maximum
increase in skin humidity was reached on day 28, with values
of 21.9 ± 1.8% and 8.9 ± 0.9% for emulsion C and control
emulsion, respectively (Fig. 3). Two days after the last appli-
cation, the increases in skin humidity were 16.9 ± 1.7% and
6.9 ± 1.0% for the two emulsions, respectively (Fig. 3),
demonstrating the sustained effect of the two emulsions on
skin hydration. As the skin hydration produced by emulsion
C was increased significantly compared with that obtained
with control emulsion, ceramides 1 and 3 in emulsion C may
cooperate synergistically to increase the water content of the
skin.
TEWL measurements
© 2008 The International Society of Dermatology International Journal of Dermatology 2008, 47, 812–819
816 Report Ceramides and skin barrier function Huang and Chang
International Journal of Dermatology 2008, 47, 812–819 © 2008 The International Society of Dermatology
Huang and Chang Ceramides and skin barrier function Report 817
Table 2 Skin erythema assays. Skin erythema (%) was expressed relative to baseline on day 0 and the untreated control
(mean ± standard deviation; n = 15; P < 0.05)
maximum decrease in TEWL was reached on day 28, with containing ceramide 3 have been reported to improve skin
values of 36.7 ± 4.7% and 5.1 ± 0.8% for emulsion C and barrier function in different conditions relative to untreated
control emulsion, respectively (Fig. 6). Two days after the last skin.16 The limited minor efficacy of the control emulsion could
application, the decreases in TEWL were 36.2 ± 4.7% and be attributed to the other lipid components, including petro-
4.5 ± 1.0% for the two emulsions, respectively (Fig. 6), indi- latum, squalene, phytosphingosine, and cholesterol. Moreover,
cating the sustained effect of the two emulsions on TEWL of the recovery of skin barrier disruption after application of the
the skin. The effect of emulsion C on TEWL of the skin is control emulsion could be a result of the occlusive effect of the
more significant than that of emulsion A or emulsion B, emulsion, which restricts water evaporation from the skin,
indicating the potential synergistic interaction between cera- thereby allowing the skin to recover. It should be emphasized
mides 1 and 3. that biophysical measurements of skin barrier function, such
as skin hydration and TEWL, are not only dependent on the
lipid composition and structure, but also on the amount of
Discussion
water and protein. Although the improvement in skin hydra-
In this study, erythema measurements performed after 1, 2, 3, tion by emulsions A and B was only slight, the decrease in
and 4 weeks of application revealed no statistically significant TEWL by these emulsions was clear and might have resulted
differences from the baseline values on day 0 or from the from the different individual ceramide subspecies which
untreated control (Table 2). Therefore, all tested emulsions, behave differently.25 Skin hydration was measured by a Cor-
including control emulsion, did not induce any visual skin neometer®825 using changes in the electrical capacitance of
irritation and were well tolerated. the stratum corneum. The electrical characteristics of the skin
Ceramides have been reported to show positive effects may be affected by various factors, including the size of the
on skin humidity and elasticity.21 In this study, emulsions corneocytes, number of cell layers in the stratum corneum,
containing ceramide 1 or ceramide 3 showed a similar effect properties of deeper skin layers, and lipid content. The
to the control emulsion on skin hydration, indicating that improvement in skin barrier function produced by emulsion
other lipid components, including petrolatum, squalene, A or B alone may be caused by the penetration of ceramide 1
phytosphingosine, and cholesterol, might show a beneficial or 3 into the stratum corneum. The lack of a clear effect of
effect on skin hydration. emulsion A or B on skin hydration might be a result of differ-
Many internal and external factors influence the properties ences in individual skin physiologic parameters.26 Interest-
of the skin, including age, genetic disposition, body region, ingly, emulsion C showed a significant beneficial effect in skin
exposure to ultraviolet (UV) radiation, and disease.22,23 The hydration, as illustrated in Fig. 3, indicating that ceramides 1
epidermis of the skin has a strong effect on the frictional and 3 contained in emulsion C may act synergistically in skin
resistance of the skin, depending on its water and lipid hydration. Certainly, other ceramides in the stratum corneum
content.24 Certainly, an increase in skin hydration and lipid are probably needed to provide the stratum corneum with a
content improves the viscous resistance against deformation. water-resistant lipid multilayer.
The results shown in Figs 1 and 2 indicate that ceramides, The results shown in Figs 4 and 5 indicate that ceramide
including ceramides 1 and 3, in stable o/w emulsions do not 1 or 3 alone may play an essential role in maintaining the
seem to play an important role in exerting a protective effect barrier function of the stratum corneum. The treatment of
on skin hydration; however, ceramide 1 has been shown to be the test sites with emulsion C was more effective in reducing
of major importance for correct stratum barrier function.15 TEWL with respect to the control area, and in comparison
The results shown in Fig. 1 indicate that ceramide 1 contained with emulsion A or B, indicating that ceramides 1 and 3 may
in emulsion A is not necessarily an essential factor for increas- work synergistically to play an essential role in the mainte-
ing skin hydration. Similarly, no significant differences were nance of the structure and water-permeability barrier func-
found in skin hydration between ceramide 3-containing tion of the skin; this indicates the existence of a correlation
emulsion B and control emulsion; however, emollients between the measured TEWL value and skin barrier function.
© 2008 The International Society of Dermatology International Journal of Dermatology 2008, 47, 812–819
818 Report Ceramides and skin barrier function Huang and Chang
It has been reported that ceramide 1 plays a dominant role in correlates of the epidermal permeability barrier. In: Elias
the organization of stratum corneum lipids.27 Moreover, PM, ed. Skin lipid. Adv Lipid Res 1991; 24: 1–26.
ceramide 3 has been shown to provide a skin barrier repair 4 Imokawa G, Abe A, Jin K et al. Decreased level of ceramides
function in a lipid-rich emollient.28 We believe that the poten- in stratum corneum of atopic dermatitis: an etiologic factor
in atopic dry skin. J Invest Dermatol 1991; 96: 523–526.
tial synergistic effects of ceramides 1 and 3 may be caused
5 Matsumoto M, Umemoto N, Sugiura H, et al. Difference in
by the formation of lateral hydrogen bonds between the head
ceramide composition between “dry” and normal skin in
groups of adjacent ceramide molecules, which reduce the
patients with atopic dermatitis. Acta Derm Venereol
permeability of skin to water, as described by Irmin and (Stockh) 1999; 79: 246–247.
Staffan.29 In addition, the existence of an attractive van der 6 Pinnagoda J, Tupker RA, Agner T et al. Occupational
Waals’ interaction between the long hydrocarbon chains guidelines for transepidermal water loss (TEWL)
of ceramides 1 and 3 may also play a potential role in the measurement. A report from the Standardization Group
synergism. of the European Society of Contact Dermatitis. Contact
To our knowledge, this is the first study to examine the Dermatitis 1990; 22: 164–178.
effect of ceramide-containing emulsions on the normal skin 7 Nilsson J. Measurement of water exchange through skin.
of Asian volunteers after a short period of application. It has Med Biol Eng Comput 1997; 15: 209–218.
8 Feingold KR, Mao-Qiang M, Menon GK, et al. Cholesterol
been shown that emulsions containing ceramide 1 or cera-
synthesis is required for cutaneous barrier function in mice.
mide 3 influence the skin barrier function of normal skin
J Clin Invest 1990; 86: 1738–1745.
as measured by TEWL. In particular, an emulsion contain-
9 Mao-Qiang M, Elias PM, Feingold KR. Fatty acids are
ing 0.2% of both ceramides 1 and 3 shows a synergistic required for epidermal permeability barrier homeostasis.
protective effect on the barrier function of normal skin. The J Clin Invest 1993; 92: 791–798.
potential mechanism of this synergistic action remains to 10 Mao-Qiang M, Feingold KR, Elias PM. Influence of
be elucidated, although suggestions have been made in the exogenous lipids on permeability barrier recovery in
previous paragraph. acetone-treated murine skin. Arch Dermatol 1993;
129: 728–738.
11 Holleran WM, Feingold KR, Man MQ, et al. Regulation of
Conclusion epidermal sphingolipid synthesis by permeability barrier
function. J Lipid Res 1991; 32: 1151–1158.
A significant positive effect on hydration and TEWL of SLS-
12 Rawlings AV. Trends in stratum corneum research and the
damaged skin was observed when the emulsion containing
management of dry skin conditions. Int J Cosmet Sci 2003;
ceramides 1 and 3 was compared with control emulsion. This
25: 63–95.
study demonstrates that ceramide 1 (0.2%, w/v) combined 13 Wertz PW, Downing DT. Acylglucosylceramides of pig
with ceramide 3 (0.2%, w/v) in an o/w emulsion (emulsion C) epidermis: structure determination. J Lipid Res 1983; 24:
shows positive synergistic effects on skin hydration and 753–758.
TEWL when applied to SLS-irritated skin. 14 Bowser PA, Nugteren DH, White RJ, et al. Identification,
isolation and characterization of epidermal lipids containing
linoleic acids. Biochim Biophys Acta 1985; 834: 419–
Acknowledgments 428.
The authors gratefully acknowledge financial support by a 15 Bouwstra JA, Gooris GS, Dubbelaar FE, et al. Role of
ceramide 1 in the molecular organization of the stratum
grant from the “MOE Guidelines for Promoting Industry–
corneum lipids. J Lipid Res 1998; 39: 186–196.
University Cooperation between Industrial Parks and Colleges
16 Kucharekova M, Schalkwijk J, Van De Kerkhof PCM,
and Universities of Technology” (private-26-resturant-011).
et al. Effect of a lipid-rich emollient containing ceramide
This study was also supported by a grant from Hungkuang 3 in experimentally induced skin barrier dysfunction.
University (HK-95-B-16). Statistical analysis by the China Medi- Contact Dermatitis 2002; 46: 331–338.
cal University Biostatistics Center is gratefully acknowledged. 17 De Paepe K, Roseeuw D, Rogiers V. Repair of acetone and
sodium lauryl sulphate-damaged human skin barrier
function using topical emulsions containing barrier lipids.
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© 2008 The International Society of Dermatology International Journal of Dermatology 2008, 47, 812–819