The effect of ceramides on skin absorption by Raman spectroscopy

Abstract

Introduction

Ceramides are essential epidermal constituents that play a critical role in skin moisturization treatment as a raw material in cosmetics formulation. Recently, ceramides have been known to be frequently applied in various cosmetic formulations. Despite ceramide's beneficial characteristics, academic research regarding ceramides and their skin absorption remains insufficient. Therefore, our study conducted clinical research employing Raman spectroscopy to investigate the effects of ceramides on skin absorption to enhance the understanding of ceramides’ dermatological functionality and their topical application in cosmetics science.

Materials and Methods

Twenty healthy individuals with dry skin have participated in this clinical trial. In this double‐arm designed trial, the test group received an investigational product with ceramides (5000 ppm) and a control group received an investigational product without the ceramides while all other components remained identical. The subjects visited the clinical research center and acclimatized for 30 min in constant humidity and temperature for equilibrium, subsequently conducting a measurement. Before the trial, the research subject's target site (lower arm area) was kept clean, devoid of any cosmetic administering 24 h before the trial when investigational product was topically applied.

Results

Our findings with Raman spectroscopy statistically demonstrate that skin absorption amount, speed and depth for both groups improved overall (p < 0.05) after administration of the investigational product. Notably, the test group received an investigational product with ceramides (5000 ppm) indicating superior effectiveness across all parameters compared to a control group from comparison analysis of each parameter (p < 0.05).

Conclusion

This study concludes that ceramide‐containing cosmetics provide a beneficial effect on skin absorption via visual and statistical results of Raman spectroscopy analysis.

1. INTRODUCTION

The popularity of cosmetic products in Korea has been surging and becoming more viral. The term “K‐beauty” is now frequently used to describe skin care products in Korea. ¹ , ² These days, there has been a notable emergence of ceramide‐containing products diversely ranging from fundamental skincare items to cleansing products reflecting a broad application of ceramide in the cosmetic industry. ³ , ⁴ , ⁵

The potential benefits of ceramide products for skin health suggest that this formulation can be a valuable approach for improving skin barrier functions and achieving depigmentation, hence ceramides are gaining much attention from Korean cosmetics industries. ⁶ , ⁷ Ceramides are crucial components of the stratum corneum, the outermost layer of the skin. ⁸ , ⁹ , ¹⁰ They play an important role in the maintenance of skin barrier functions by preventing skin dryness, agent for depigmentation, and providing protection from external damages. ⁶ , ¹¹ , ¹² Due to its characteristics, topical administering products with ceramide that resemble physiological skin are found to enhance skin barrier functions. ¹³ These formulations help replenish ceramide levels in the skin, thereby keeping water permeability and decreasing transepidermal water loss (TEWL) overall maintaining human physiological homeostasis. ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² These potential benefits of ceramide products have triggered significant attention from the cosmetics industry. ³ , ⁴ , ⁵

However, the extraction and purification of ceramides, as well as the development of ceramide‐base products remains a challenge due to the difficulty in precisely identifying ceramides. ¹⁴ , ¹⁵ , ¹⁶ Therefore, manufacturing effective products in terms of skin hydration, absorption, and safe patient compliance varies depending on the competence of manufacturers and suppliers. Despite the limited commercial availability of ceramide products, the industry is investing great effort due to the outstanding benefits of skin hydration. ¹³ , ¹⁵ , ¹⁷ , ¹⁸ Nonetheless, there is a lack of research on ceramides and their skin absorption potential with visual data.

Raman spectroscopy is regarded as a versatile tool that utilizes different frequencies of light scatter at the spectra, and it is able to activate specific molecular vibration and rotation throughout the skin; it is non‐invasive, non‐destructive, and real‐time. ¹⁹ , ²⁰ , ²¹ Therefore, Raman spectroscopy facilitates in vivo testing of ceramide permeability and efficacy by using scattered light spectra at different frequencies to reveal molecular vibrations and rotation. ²¹ , ²² , ²³ This enables the analysis of skin penetration of ceramide and its permeability in vivo. ¹⁹ , ²⁰ , ²¹ Raman spectroscopy is used in a wide range of scientific fields including dermatology and pharmaceuticals, ensuring cost savings and product quality assurance. ²¹ , ²² , ²⁴ Raman spectroscopy has shown efficacy as an analysis method investigating skin moisture effect by examining the peak of water molecules and other related components. ²⁰ , ²⁵

In the past, Raman spectroscopy relied solely on the identification of molecular peaks for confirming results purposes. ²³ However, advancements in technology enabled enhanced versions of Raman spectroscopy imaging with intricate 2D and 3D visualizations along with statistical peaks and values. ²⁶ , ²⁷ This capability not only allows improved comprehension of the research data in depth but also facilitates the wider application of this technique such as advertising specific cosmetic prototypes. This study is undertaken to evaluate the differential skin absorption properties depending on cosmetic formulations with and without ceramides via Raman spectroscopy. We aim to provide meaningful advancement to technology development in the cosmetic industry.

2. MATERIALS AND METHODS

2.1. Investigational product (IP)

“irecipe Aqua Phytoplex Cera Cream (Sunjin Beauty Science Co., Ltd., Seoul, Korea)” was used as a skin moisturizer and Investigational Product (IP) in this study. The test group received IP consisting of ceramide (5000 ppm), while the control group received IP with identical composition but devoid of ceramide. The IP of the test group is regarded as a notable product in the Korean cosmetic market for its ceramide moisturizer efficacy. The IP was topically applied for each of the two groups on the subject's lower arm area after acclimatization in a controlled environment. Raman spectroscopy was subsequently utilized to evaluate relevant parameters of skin absorption in this study.

2.2. Ethical approval (IRB)

This clinical research was approved by the Institutional Review Board of Sunjin Beauty Science Co., Ltd.. on July 01, 2024. The clinical research was conducted according to the Declaration of Helsinki and GCP Guidelines (ICH E6 [R1, R2]). The IRB approval number for this study is “SCRC_IRB_24_IR0005_0007.”

2.3. Subjects

Twenty healthy Korean individuals including both males and females, with dry skin were recruited for this clinical study. The research subject had no history of supplementation or medication targeting skin moisture conditions.

2.4. Environmental condition and measurement

Before measurement, the subject acclimated for 30 min in a controlled environment. The condition was maintained with no air movement or direct sunlight exposure. The temperature ranged from 20°C to 24°C, and relative humidity between 40% and 60% to achieve equilibrium. The IP's duration of action is 30 min after it is administered. Therefore, the measurements were recorded after the IP administration, following a 30 min acclimatization under the same initially described conditions.

2.5. Skin absorption measurements via Raman spectroscopy

Evaluation of skin absorption on the subject's lower arm was acquired utilizing a Nanofinder_FelxG Raman Spectroscopy (Tokyo Instrument, Tokyo, Japan). Raman spectroscopy is a non‐invasive device that confirms the skin absorption of substances by measuring the specific wavelength of laser light. ²⁰ , ²³ , ²⁸ Our research instrument operates on the principle of the Raman scattering phenomenon of light, detecting and quantifying fluctuations of vibrational energy before and after the collision of light and matter. ¹⁹ , ^{22 . 23}

This device was used to evaluate three parameters of skin absorption amount, speed, and depth of the subject after applying IP. These evaluations were conducted without interrupting the skin by 785 nm laser irradiation and analyzing the Raman spectrum of a specific wavelength area. The maximum measurement depth is 70 μm and the interval (X‐axis, depth) of 5 μm. A dose amount of 20 μL of the IP was topically applied at the target site (lower arm area) of the research subject. Two measurements were obtained, one before the application and one after the IP's duration of action (30 min). Skin absorption properties were analyzed based on three parameters. Skin absorption amount (expressed as Arbitrary Unit, A.U.) indicates the relative amount of investigational product absorbed into the skin after the duration of action. Skin absorption speed (μm/h) indicates the depth of skin penetration by IP per unit time. Skin absorption depth (μm) is the maximum depth of IP absorption, measured before and 30 min after IP application. The mean value at each measurement was calculated as mean ± standard deviation (SD). The change amount was calculated accordingly.

2.6. Statistical analysis

Data analysis and acquisition were performed with SPSS Statistics Software ver. 29 (IBM Corp., Arming, NY), and all the collected data is represented by mean ± standard deviation. The normality was assessed by the Sapiro–Wilk test. Statistical probability was confirmed at a 95% confidence level with p < 0.05. Therefore, for a statistical comparison regarding the effect of the investigational product use before and after IP administration, a non‐parametric (Wilcoxon signed‐rank test) method was solely used rather than using parametric (paired t‐test) method to. In addition, the differences between the test group and the control group were analyzed via calculating the changes within each group. Similarly, only one statistical analysis was conducted—a non‐parametric (Mann–Whitney U‐test) method, excluding the parametric method (independent t‐test for each group).

3. RESULTS

The clinical trial data of this study were obtained from 20 healthy participants aged between 20 and 37 years (mean age: 27.75$\pm$4.58 years) with dry skin, all of whom agreed and signed the informed consent. The skin absorption properties affected by the presence of ceramide in the product were analyzed using the Nanofinder_FlexG Raman Spectroscopy instrument (Figure 1). The results were obtained based on three designated parameters, skin absorption amount (A.U.), skin absorption speed (μm/h), and skin absorption depth (μm).

FIGURE 1.

Experimental setup of Nanofinder FlexG Raman Spectroscopy, Tokyo Instrument, Tokyo Japan for data acquisition on the research subject. Measurement parameters include skin absorption amount (expressed as Arbitrary Units, A.U), speed (expressed in μm/h), and depth (expressed as μm) in SI units.

Two treatments of arms, the test group with ceramide‐containing IP and the control group with an identical component of IP excluding ceramide were assessed on skin absorption parameters before and after the topical administration was collected in our clinical trials. As depicted in Figure 2, a comparative analysis of this double‐arm trial revealed evident changes across all of the skin absorption relevant parameters for both groups (Figure 2). Table 1 shows each group's calculated average value and standard deviation and time points in measured skin absorption properties. In addition, the p‐value from each group indicates statistically significant changes across all parameters (Table 1).

FIGURE 2.

Effect of Investigational product on three skin absorption parameters between the control group and the test group before and 30 min after product administration (mean ± standard deviation; n = 20). *denotes significant changes at p < 0.05 compared between two groups, calculated using the Wilcoxon signed‐rank test method.

TABLE 1.

Mean, standard deviation (S.D.), and p‐value of the control group and test group before and after product administration (20 number of research subject).

Parameters	Group	Time points	Mean ± S.D.	p‐value
Skin absorption amount (A.U.)	Control group	Before	327,296.8 ± 4289.7	<0.05 *
		30 minu after use	495,433.2 ± 110,293.0
	Test group	Before	330,196.3 ± 7325.2	<0.05 *
		30 min after use	617,029.4 ± 49,606.2
Skin absorption speed (μm/h)	Control group	Before	70.5 ± 7.5	<0.05 *
		30 min after use	119.3 ± 24.2
	Test group	Before	69.2 ± 4.3	<0.05 *
		30 min after use	138.3 ± 5.8
Skin absorption depth (μm)	Control group	Before	35.2 ± 3.8	<0.05 *
		30 min after use	59.7 ± 12.1
	Test group	Before	34.6 ± 2.2	<0.05 *
		30 min after use	69.2 ± 2.9

^*Significantly different at p < 0.05 compared between time points by Wilcoxon signed rank test.

Figure 3 illustrates the statistical result of a comparative analysis between the test group and control group regarding the change amount in improvements among three skin absorption parameters within each group. According to our result, the test group that received the ceramide‐containing IP showed superior efficacy and statistically significant result in enhancing skin absorption amount, depth, and speed compared to the control group devoid of ceramide. The test group resulted relatively greater in change amount in those parameters (p < 0.05) (Figure 3 and Table 2).

FIGURE 3.

The changes within each group for three skin absorption parameters before and 30 min after product administration, indicating differences in change between the test group and the control group (mean$\pm$standard deviation; n = 20). †denotes significant changes at p < 0.05 compared between two groups, calculated using the Mann–Whitney U‐test method.

TABLE 2.

Change amount and p‐value of skin absorption parameters and, skin absorption amount (A.U.), speed (μm/h) and depth (μm) within each group.

Parameters	Group	Change amount	p‐value
$\mathrm{\Delta }$Skin absorption amount (A.U.)	Control group	168,136.4 ± 109,107.4	<0.05 *
	Test group	288,745.3 ± 49,680.6	<0.05 *
$\mathrm{\Delta }$Skin absorption speed (μm/h)	Control group	48.9 ± 26.4	<0.05 *
	Test group	69.2 ± 6.9	<0.05 *
$\mathrm{\Delta }$Skin absorption depth (μm)	Control group	26.1 ± 12.6	<0.05 *
	Test group	34.6 ± 3.5	<0.05 *

^*Significantly different at p < 0.05 compared between groups by Mann–Whitney U‐test.

The clinical data of each research subject were initially expressed as 2D images in values using imaging software. The skin absorption amount and depth were derived under 2D images. Skin absorption speed was calculated by incorporating the application time as a variable input (Figure 4). The multiple 2D images derived from the trials were automatically merged into 3D images via Raman spectroscopy software, providing a pillar‐shaped, three‐dimensional representation within the visible spectrum of electromagnetic wavelengths (380–740 nm) (Figure 5). Figures 4 and 5 facilitate the evaluation of differences in skin absorption changes regarding the presence of ceramide in IP. No adverse events or side effects were reported by the 20 study participants during and after the completion of this trial.

FIGURE 4.

In vivo Raman spectra, spatial distribution of ceramide penetration by investigational product in the subject's skin before and 30 min after product administration. Compared between the control group and the test group, visualized in two dimension, x and y plane. (Visible spectrum λ = 380–740 nm).

FIGURE 5.

In vivo Raman spectra, spatial distribution of ceramide penetration by investigational product in the subject's skin before and 30 min after product administration. Compared between the control group and the test group, visualized in three dimension, x, y, and z plane (visible spectrum λ = 380–740 nm).

4. DISCUSSION

This study evaluates a numerical and visual variation of changes in skin absorption parameters depending on the presence of ceramides in IP using Raman spectroscopy. It has been reported that people with chronic dry skin tend to prefer cosmetic products with high efficacy in skin absorption. Therefore, this study investigates ceramide‐containing cosmetic formulations that have beneficial effects on skin absorption properties. To confirm the validity of experimental results, all the participants in the research subject were adult individuals with dry skin but no background of skin‐related diseases.

Ceramides are major phospholipids that compose 40%–50% of the outer layer of skin, the epidermis. ⁸ , ¹¹ , ²⁹ It is known to maintain the water permeability barrier of skin thus providing healthy and hydrated skin by supporting the skin's natural barrier formation and decreasing TEWL. ⁹ , ¹⁸ , ³⁰ Studies have also reported that ceramide prevents the entry of germs, inhibiting inflammation and restoring damage. ⁶ , ¹² Ceramides absorbed into the body from external sources such as ceramides supplementation enhance skin hydration thus preventing skin dryness. ⁸ , ⁹ , ¹³ , ³⁰ Ceramide deficiency triggers increased TEWL and is known to cause various skin disorders such as atopic dermatitis due to weakened skin barrier function. ¹⁰ , ¹⁸ Many cosmetic companies are investing their work to manufacture and develop topical formulations that contain high ceramide content due to their prominent potential. ⁵ , ¹⁵ , ¹⁷

However, the commercial availability of ceramide as a cosmetic raw material is limited, primarily due to its high market price. ¹³ , ¹⁸ Additionally, ceramide's highly hydrophobic properties and lack of technological advancements hinder its incorporation into cosmetic products. ¹¹ , ¹⁴ , ¹⁵ , ¹⁶ The IP used for the test group in this clinical trial contains 5000 ppm of ceramide.

Raman spectroscopy measurements approach are applied in various industrial fields such as life sciences, pharmaceuticals, and geological research for numerous purposes. ²¹ , ²² , ²⁴ In this study, Raman spectroscopy was employed as a effective tool for monitoring specific bimolecular changes of physiological skin in real‐time, non‐invasively, and non‐destructively. ¹⁹ , ^{20 ,} Raman spectroscopy provided statistical analysis data as well as 2D and 3D visualizations. Historically, Raman spectroscopy was only limited to confirming the presence of specific molecular wavelengths in chemical compounds. ²³ However, as technology develops 3D Raman spectroscopy now allows effective observation of visual changes in comprehensive layers of skin and investigation on the absorption properties of IP. ²² , ²⁵ , ²⁶ , ²⁷ Our study has utilized this technology in our research method to verify relative differences in skin absorption properties of cosmetic product (IP) depending on the presence of ceramide. Our methodological approach will enable other fields of cosmetic industries to conduct a resemble‐research to investigate how the presence of certain substances in their products influence certain physiological properties, extending beyond not only limited to ceramide.

5. CONCLUSION

Our findings revealed that IP consisting of ceramide for the test group (5000 ppm) and IP without ceramide for the control group both indicated overall improvement across all three parameters of skin absorption amount, skin absorption depth and skin absorption speed when the product was topically administered to the target area of the research subject. Besides, the test group showed statistically valid outstanding effectiveness in changes across all parameters compared to the control group.

This study illustrates that ceramide‐containing products enhance skin penetration into the epidermis according to the results of three parameters of skin absorption, amount, depth, and speed. Therefore, for individuals with dry skin or seeking cosmetics with superior absorption efficacy, it is recommended to use a ceramide‐containing formulation. This study result shows the presence of ceramide significantly improves and exerts a beneficial effect on three parameters with respect to skin absorption.

The measurement methodological approach of our study provides analysis of IP distribution across a wide range of planar dimensions (2D, 3D), also complemented by statistical results facilitating accessibility and understanding of our result for a wider audience. This study suggests that when investigating specific components of prototypes or final manufactured cosmetic products, the application of Raman spectroscopy instrument analysis can provide precise validation of results.

In conclusion, our study demonstrates that ceramide‐containing cosmetic product achieves more rapid, deeper and greater absorption into the skin, as suggested by both numerical and visual data obtained via Raman spectroscopy technology.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Kim SH, Yun J, Kim W, et al. The effect of ceramides on skin absorption by Raman spectroscopy. Skin Res Technol. 2024;30:e70046. 10.1111/srt.70046

DATA AVAILABILITY STATEMENT

Data derived from our own clinical research.