Arginine–fructose–glucose from red ginseng extract reduces stiffness of keratin fiber in corneocyte of skin

Abstract

Purpose

The moisture content of the stratum corneum of the skin changes depending on the external environment. The structure of keratinous fiber protein in corneocyte of the skin changes depending on the amount of moisture. As the moisture decreases, the population of the alpha‐helix increases, the beta‐sheet deceases, and the stiffness increases accordingly. Here, we investigated the effect of humectants from ginseng on the keratin structure.

Methods

Corneocyte was prepared from dry porcine skin with disc tape and measured through ATR‐FT‐IR. The signal from amide I of the keratin protein in corneocyte was detected, and the change in the ratio of alpha‐helix and beta‐sheet was calculated. The test samples were treated on the exfoliated corneocyte, and the degree of change was checked.

Result

Arginine–fructose–glucose (AFG)‐enriched extract of red ginseng was effective in changing the keratin structure and was superior to humectants such as glycerin. However, arginine, mono sugar were not effective, and the AFG form in which two sugars were bound to one amino acid could perform its function.

Conclusion

The present study suggests that AFG, when applied to cosmetics, is expected to improve skin texture in a different way from existing moisturizers represented by glycerin by reducing the alpha‐helix structure of corneocyte keratin.

1. INTRODUCTION

The skin consists of the outer epidermis and the inner dermis. The epidermis serves to prevent the invasion of foreign substances from outside and to prevent the loss of moisture inside. The epidermis differentiated from the basal layer forms the stratum corneum. The stratum corneum consists of corneocytes and intercellular lipid layers. This structure is expressed as a brick‐and‐mortar model. ¹ The corneocytes occupy most of the stratum corneum area and volume and are composed of keratin fiber proteins and natural moisturizing factor (NMF) components. ²

The stratum corneum is the only structure in which the moisture content changes depending on the external humidity. ³ The structure of the corneocyte changes according to the moisture change in the stratum corneum. ⁴ If it contains moisture, the volume may expand up to four times. In addition, the structure of keratin also changes between the alpha‐helix structure and the beta‐sheet according to the change of the moisture content. ⁴ , ⁵ When water is insufficient, the ratio of alpha‐helix increases, and stiffness also increases. ⁶

External treatments such as cosmetics form an oil film on the surface of the skin to suppress the loss of moisture and provide moisture ⁷ and humectant ingredients to maintain moisture in stratum corneum layer. Glycerin and polyol components have been most commonly used. ⁸ In order to analyze the moisture content in the stratum corneum, the method as electrical conductivity was used, and it was used as a method to identify the moisturizing function of cosmetics. ⁹

Meanwhile, the change in stiffness of keratin due to drying appears as a feeling of roughness and pulling of the skin. ¹⁰ Therefore, it is important to directly confirm the change in the keratin structure. ³ However, indirect analysis by electrical conductivity cannot directly confirm the change of keratin structure. An efficient way to view this is to analyze the amide I peak through FT‐IR. ⁴ Through this, it is possible to confirm the change of keratin in the skin corneocyte in a noninvasive manner (Figure 1A).

FIGURE 1.

Analysis of keratin stiffness in stratum corneum using ATR‐FTIR spectroscopy ¹⁶ : (A) Stiffness changes according to the structural change of the keratin protein constituting the stratum corneum; (B) occlusive or humectant inhibits the evaporation of water from the inside of the skin; and (C) structural changes that occur by penetrating into the excised stratum corneum are observed.

There are various non‐saponin‐based ingredients in ginseng, sugar components such as glucose and fructose, and amino acids. In the process of manufacturing red ginseng, the bond between the sugar component and the amino acid component occurs due to heat, and arginine–fructose–glucose (AFG) is produced. So, AFG‐enriched extract of red ginseng could be produced. ¹¹ Studies on various efficacy of AFG‐enriched extract were conducted, and physiological functions for circulatory system diseases and immunity enhancement have been reported. ¹²

In this study, we tried to confirm the effect of AFG‐enriched fraction on the skin, and the study was started in terms of physical changes rather than physiological changes. As a result, it was confirmed that the stiffness of the stratum corneum keratin can be improved by the AFG‐enriched fraction. In addition, it was confirmed that fructose and glucose or arginine itself did not show any efficacy, and only the AFG structure performed its function. This implies the importance of research on non‐saponin‐based ingredients of red ginseng and the possibility of application in cosmetics.

2. MATERIALS AND METHODS

2.1. Materials

AFG (Ambo institute, Korea); AFG‐enriched extract (Korea Ginseng Corporation; KGC, Korea); glycerol, fructose, maltose, and glucose (Sigma, USA); SC tape (D‐Squame Sampling disc, 22 mm diameter, CuDerm, USA); porcine skin (1 mm thickness, APURES, Korea) were purchased. ART‐FT‐IR (Jasco 4200 [JASCO, Japan] and Corneometer [Howskin, Korea]) were used.

2.2. Sample treatment

Porcine skins were dried in 32°C chamber for 6 h. Tape stripping (D‐Squame Sampling Disc, 22 mm diameter, CuDerm) was performed on the surface of dried porcine skin to collect SC samples, and 200 μl of a test sample was dropped at the center of the SC tape and incubated for 4 h. The incubated samples were then washed with distilled water and dried by air blowing.

2.3. Determination of stiffness using keratin configuration analysis

Infrared amide I band was analyzed on the SC tape using FT‐IR. The amide I band (1600–1720 cm⁻¹) of the FT‐IR spectrum of SC was then resolved. The band was centered at 1651 cm⁻¹, which indicated the vibration spectrum of a polypeptide in the α‐helix conformation, whereas the other bands at 1631 cm⁻¹ indicated the vibration spectrum of a polypeptide chain in the β‐sheet conformation. An increased proportion of keratin in the β‐sheet conformation was assumed to indicate a reduction in stiffness.

2.4. Statistical processing

All samples were measured with five replicates, followed by the mean and standard deviation. Only the significant difference between the two groups was analyzed, and **p < 0.05 was indicated after the statistical verification of the student's t‐test (EXCEL, USA).

3. RESULTS

3.1. The relationship between the moisture content of keratin and the degree of keratinization

Porcine skin was dried according to the incubation time in a 32‐degree chamber. After measuring the moisture content of the skin over time (Figure 2A), the keratin of the skin was stripped with a tape and analyzed using FT‐IR. The initial moisture content of 37.50% ± 2.51% of porcine skin decreased to 30.39% ± 1.9 8% and 26.36% ± 3.93 % at 2 and 4 h (Figure 2, respectively. For each dried sample, the peak in the FT‐IR band of amide I was convoluted. After that, the height of 1651 cm⁻¹ peak (alpha‐helix) was divided by the value of 1631 cm⁻¹ peak (beta‐sheet) to calculate the degree of keratin stiffness before sample treatment. The alpha/beta ratio was increased from 1.36 ± 0.03 initially to 1.55 ± 0.03 and 1.69 ± 0.09 as the water content decreased (Figure 2).

FIGURE 2.

Representative FT‐IR spectrum of tape‐stripped stratum corneum obtained whole porcine skin according to drying time: (A) Changes in humidity are measured for the whole skin according to drying time; (B and C) according to the drying time, the ratio of beta‐sheet at 1636 to alpha at 1651 in amide I was calculated by analyzing FT‐IR on the tape‐stripped stratum corneum.

3.2. Effect of arginine–fructose–glucose (AFG)‐enriched extract on keratin stiffness

To confirm the effect of AFG‐enriched extract on keratin stiffness of corneocyte (Figure 3C), it was compared with glycerin, the most frequently used humectant. For this, the porcine skin was dried in a 32‐degree chamber for 6 h. The stratum corneum of porcine was removed using SC tape. Their degree of keratin stiffness before sample treatment was first analyzed using FT‐IR. Absorbance data in the area of 1600–1680 cm⁻¹ were obtained, and the size of the intrinsic peaks was calculated using the Gaussian fitting. After that, the height of 1651 cm⁻¹ peak (alpha‐helix) was divided by the value of 1631 cm⁻¹ peak (beta‐sheet) to calculate the degree of keratin stiffness before sample treatment.

FIGURE 3.

FT‐IR analyzes the recovery of keratin stiffness according to various formulations: (A and B) After treating various samples on the tape‐stripped stratum corneum, the ratio of alpha‐helix and beta‐sheet in the keratin amide I band was measured; (C) arginine–fructose–glucose (AFG) generated during the production process of red ginseng.

After that, about 300 μl of samples of distilled water, 10% glycerin in water, and 5% AFG‐enriched extract in water were treated on SC tape in a 32‐degree incubator for 4 h. After that, the reaction was terminated by washing with distilled water and drying with an air blow method. For these samples, FT‐IR data was obtained in the same manner, and the degree of keratin hardening after sample treatment was calculated. Finally, changes in the degree of stiffness before and after sample treatment were confirmed. As a result, in the case of the distilled water treatment group, the degree of stiffness increased by 23.26% ± 16.85%. In contrast, in the case of 10% glycerin, the degree of keratin stiffness increased by 14.55% ± 13.75% after sample treatment. However, in the case of AFG extract, it decreased by 14% ± 4.00% (Figure 3A,B).

In the corneocyte, not only keratin fibers, but also various NMF components are present. Their interaction with keratin fibers can affect the morphology of keratin proteins. In this experiment, when distilled water was treated, the degree of stiffness increased, and it is assumed that the degree of stiffness increased due to the extraction of these NMF components by distilled water. At this time, when components such as glycerin are present, the increase in stiffness degree can be slightly reduced. In contrast, when the AFG‐enriched extract was treated, it was confirmed that the degree of stiffness decreased compared to before treatment.

3.3. The structure of AFG affects the improvement of keratin stiffness

AFG‐enriched extract contains about 15% of AFG, making it the largest percentage. Therefore, in order to confirm whether the component that actually shows efficacy among the components is AFG, the function was confirmed using a single component of AFG. In addition, arginine‐fructose (AF), AFG, and arginine were prepared as 5% samples in order to confirm the important part of the efficacy in the structure of AFG. In this study, the stiffness was increased after the treatment of arginine with 151.1% ± 38.24% and AF with 126.6% ± 18.75%. However, the AFG component decreased stiffness to 75.6% ± 13.90%. From this, it was confirmed that AFG itself has a function in improving keratin stiffness (Figure 4).

FIGURE 4.

FT‐IR analyzes the recovery of keratin stiffness according to various arginine–fructose–glucose (AFG) components: (A) structures of each AFG component; (B) after treating various samples on the tape‐stripped stratum corneum, the ratio of alpha‐helix and beta‐sheet in the keratin amide I band was measured.

In addition, arginine itself resulted in an increase in the degree of stiffness; however, AF and AFG in which sugars were additionally bound to amino acid were assumed to gradually decrease the degree of hardening. From this, the formation and accumulation of AFG in the manufacturing process of red ginseng is expected to be an important process that makes the key component of hardening improvement.

3.4. Comparison of keratin stiffness improvement between sugars and AFG

The arginine component did not improve the degree of stiffness, and binding of sugar components appeared to be important in improving the degree of hardening. So, we tried to confirm whether the use of the sugar component itself would show a better function than the use of AFG. Monosaccharides, such as glucose and fructose, and maltose similar to the structure of sugars bound to AFG were used as disaccharide components. These components and AFG were made into a 5% solution and used in the experiment (Figure 5A). As a result, the monosaccharide component did not show any significant improvement. However, maltose, a disaccharide, has improved hardenability compared to before use with 89.24% ± 13.38% and showed superior function compared to glycerin (p < 0.05). In this condition, the degree of hardening of AFG decreased after use, resulting in 75.6% ± 12.2%. At this time, it was confirmed that it showed a better function than maltose (p < 0.05) (Figure 5B). From this result, it can be seen that the AFG structure formed by the combination of arginine and disaccharide is important to function.

FIGURE 5.

FT‐IR analyzes the recovery of keratin stiffness according to various sugar components: (A) structures of each arginine–fructose–glucose (AFG) component; (B) after treating various samples on the tape‐stripped stratum corneum, the ratio of alpha‐helix and beta‐sheet in the keratin amide I band was measured.

4. DISCUSSION

Maintaining moisture in the skin is an important factor in maintaining healthy skin. But dry skin is common during the cold, dry winter months and becomes more prevalent with age. ¹³ Several inflammatory skin conditions, such as atopic dermatitis and irritant contact dermatitis, cause localized areas of dry skin also. Glycerin is a well‐known humectant that has been included in moisturizers to improve. Furthermore, polyol like butylene glycol, dipropylene glycol, organic acids like lactate, urea, and amino acid are used in a number of moisturizer.

The dryness and stiffness of the skin is one of the uncomfortable feelings caused by dehydration, which is due to the change in the structure of keratin. Verification of the change in keratin structure caused by the supply of moisture is an important index that can confirm the improvement of stiffness, instead of an indirect method of measuring electrical conductivity.

Conventional humectants work by holding moisture around their molecular structure. Therefore, in the case of the porcine skin installed in the Franz cell, it is thought that the stiffness of the keratin will decrease if humectants catch the evaporated moisture from lower side skin well (Figure 1. However, in this study, we tried to confirm the ability to change the structure by penetrating into keratin on the stripped tape from the upper side (Figure 1C). This method is thought to mimic the situation in which moisture penetrates into the skin during the treatment of external treatments. It was confirmed that AFG generated during the manufacturing process of red ginseng significantly changed the degree of hardness compared to glycerin in penetration condition. It was also confirmed that the sugar structure bound to arginine plays an important role.

The interaction between the sugar component and the protein is well known for the study of the protective action of the protein under dehydration conditions of the protein. Most common protein degradation process are denaturation and non‐covalent aggregation. This can be caused by various stresses like heat and dehydration. In this case, sugar is used as stabilizer against dehydration stress. The water replacement theory describes that during drying, the hydroxyl group of sugar bonds with the proteins, replacing hydrogen bond between eater and the protein. ¹⁴ So, the protein's original conformation is maintained. With a similar mechanism, it can be assumed that the sugar structure may help restore the alpha‐helical structure of keratin from dehydration process.

Meanwhile, the effect of amino acids such as arginine on the structure of keratin is well known in the field of hair care. Like the skin, the structure of the hair is made up of keratin. The ability of keratin proteins and peptides of different origins to restore hair structure and mechanical properties was investigated. In these studies, it is suggested that the cationic arginine binds to negatively charged keratin fiber and improve the restore function of protein extraction. ¹⁵ In this study, arginine itself did not improve the degree of hardening, but the more the sugar component was incorporated, the better the performance of improving the degree of hardening. Moreover, arginine‐coupled form was better than disaccharide. Through this, it can be assumed that the arginine component delivers sugar components to keratin better, thereby helping to improve the intensity of the stiffness.

Through this study, it was confirmed that AFG produced by the Maillard reaction between maltose or glucose and arginine during the manufacturing process of red ginseng¹² (Figure 3C) is an excellent ingredient in improving the hardening of keratin. It is expected to be applied to various cosmetics formulations in the future and used as an ingredient that can complement existing moisturizers.

CONFLICT OF INTEREST

The authors state no conflict of interest to declare.

Kim S, Lee SH, Lee S‐H. Arginine–fructose–glucose from red ginseng extract reduces stiffness of keratin fiber in corneocyte of skin. Skin Res Technol. 2023;29:1–7. 10.1111/srt.13217

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.