Carole Gherardi (Dupont de nemours)
Piera Peric (Dupont de Nemours Deutschland)
Hair fibres have no active or renewable protective mechanisms, yet hair provides an essential beauty and protective function where each fibre may need to last several years. Glycine-betaine (GENENCARE® OSMS BA by DuPont Industrial Biosciences), well known for its protein protection and osmolyte properties has established skin benefits and has the potential to protect and improve the protein structure of hair as it experiences the rigours of everyday cosmetic routines. Using a variety of analytical HPLC and HPDSCHigh Pressure Differential Scanning Calorimetry, molecule vibration and rotation (Raman spectroscopy) and mechanical (tensile testing techniques), it has been demonstrated that Glycine-betaine is a natural component of human hair. In addition, it has been shown that it can be delivered to the cortex of the hair using typical hair care formulations. When present in the cortex, Glycine-betaine helps to influence the relationship between intermediate filamentsIF’s and intermediate filament associated proteins IFAP’s resulting in improved hair strength.
Hair is functional and essential to life. However, the fully formed fibre does not have any metabolic activity nor the self-repair system. All functional controls of protein and membrane results from the final composition and internal arrangement of proteins and lipids, and these are determined within the hair follicles. Therefore, it is assumed that hair contains numerous internal components and chemical reactions or mechanisms in order to maintain the protein structure and function of hair, in various general environmental settings, daily consumer habits and rigor of behavior. When considering each fibre, the hair must maintain this structure and function for up to 3 years equally in the long hairstyle.
The glycine-betaine (INCI name Betaine) is made as an osmotic inhibitor that protects cells and proteins from heat and oxidative stress. These extreme conditions are strongly related to the daily use of cosmetics, such as shampooing, bleaching, dyeing, perming, and heat drying. In fact, in an environment with low humidity and additional heat treatment and surfactants, which are known to affect hair structure and function and cause denaturation, the range of total saturation will be wide. Glycine-betaine is extracted by chromatography from Sugar beet molasse. This could reduce the protein damage and skin irritation caused by surfactant. It also appeared to increase moisturization of the skin. All of these effects are consistent with the function of osmotic inhibitors. Therefore, we hypothesized that glycine-betaine naturally exists in the hair, forms a natural protection mechanism and its supplementation by hair care products have noticeable effects on hair.
It is ideally suited for penetrating hair when it is washed away with a small (molecular weight 117) water soluble molecule and delivered with the material left. To explain the hypothesis, we need to demonstrate the presence of fibres, the delivery to inside of hair, the activity in the hair cortex, and finally the noticeable effects of hair. For this, we used the following techniques to demonstrate the effects of glycine-betaine on hair.
Glycine-betaine, the natural component of hair
We used the HPLC technique for the glycine-betaine analysis. Through hot water extraction,we found a noticeable level of Betaine in the hair. 
Delivery of glycine-betaine from shampoo in vivo
The hypothesis has been proved by showing that washing out hair with products such as shampoo containing glycine-betaine could add glycine-betaine to fibres.
The hair samples from 10 individuals for in vivo study, the subjects did not use or eat the products containing glycine-betaine. As their general hair care regimen, everyone was given a shampoo containing 5% glycine-betaine for random use. New samples were taken from each individual after 6 weeks and prepared as above. Glycine-betaine significantly increased 7.9 times (Fig. 1).
Penetration of glycine-betaine into the fiber substructure
Hair is a complex, multi-compartment substrate (cuticle, cortex, medulla, CMC, etc.) that is often challenging to draw definitive conclusions in the study of ingredient utilization. A large amount of sample analysis does not distinguish between surface deposition and penetration, and it is often difficult to grasp where the material is . Therefore, additional methods to identify the location of the material and the action site should be used. Using Raman confocal microscopy, it was found that glycine-betaine penetrates deeply into the wool fibres; it is a fibre having a structure with permeability characteristics very similar to that of hair. The results show that glycine-betaine exists in the cuticle and cortex and has penetrated into the center of the fibre (Fig. 2).
The results using fluorescence microscopy (Fig. 3) suggest that glycine-betaine penetrates into the natural and bleached cortex from existing shampoos.  Although many ingredients can penetrate hair fibres, it is necessary to demonstrate the effect on the protein structure and the resulting hair characteristics. There is almost no tensile properties of fibres, or it is determined by the cortex which is not or little involved with the cuticle. The cortex consists of many long cortical cells.
Each cell is contained in a matrix of proteins related to the intermediate fibres (Fig. 4). And, it contains many intermediate fibres which are well known as the two-phase model proposed by Feughelman.  For example, many processes such as surfactants, bleaching, heat, etc. can affect IF's and IFAP's interaction, and can negatively affect hair strength and performance.
Measurement of the effect of glycine-betaine on cortical proteins
Protein structure and relationship can be identified using differential calorimetry . This method generally reports two values: the TdDenaturation temperature, in other words, the denaturation of the intermediate fiber protein and the enthalpy (ΔH) which measures IFAP's or viscosity of IF environments. In the following study, glycine-betaine was applied to normal hair and bleached hair with 2% aqueous solution for 2 minutes, and the effect on denaturation of Td and enthalpy was measured. Glycine-betaine had little effect on Td, however the enthalpy value was reduced, especially in the normal hair (Fig. 5). These data clearly demonstrate the presence of glycine-betaine in cortical cells and that how it affects the interactions between intermediate fibres and their associated proteins. These studies show that glycine-betaine can be transferred from the Rinse-off products to the hair cortex, and the effect was measured in the essential protein structure of the fibres. However, it is questionable whether the effects of glycine-betaine can demonstrate most of the hair characteristics.
Effects of glycine-betaine on hair tensile properties (strength)
Tensile testing has long been established method and is designed to measure changes in the longitudinal properties of fibres. Load elongation is controlled by the hair cortex, which has no relation to the cuticle.  We applied the 5% aqueous solution of glycine-betaine to normal Asian hair for 15 minutes. Tensile data showed an increase in elastic modulus and elastic stress at 15% elongation index (Fig. 6). Two measurements demonstrate that glycine-betaine directly affects the cortical properties, resulting in an increase in hair strength. In both measurements, the bending stiffness increased. As a result, it can be expected that the increased elastic coefficients will consequently improve the body and volume of the hair bundle or hair style.
To summarize, glycine-betaine is an ideally suitable small and natural ingredient for delivering moisture to hair. Glycine-betaine appeared to be associated with the natural protection mechanism of fibres and could increase throughout the fibres. As a result of analysis, glycine-betaine penetrated the hair cortex and affected the interactions between intermediate fibres and intermediate fibre-related proteins. It is well known that all hair (natural hair and treated hair) have fiber damage due to steady decrease in mechanical strength from root to end. Maintaining fibre strength allows consumers to keep longer and better hair, hairstyle and length. As a result, hair in the better condition can maintain its appearance, body, and shape. This will also reduce the likelihood of damage and increase consumer satisfaction.
 Pulliainen, K, Nevalainen H, Väkeväinen H, Jutila K, Gummer C.L., An analytical method for the determination of betaine (trimethylglycine) from hair. International Journal of Cosmetic Science, 2009, Volume 32 (2),135–138
 Gummer C.L., Elucidating penetration pathways into the hair fiber using novel microscopic techniques J Cosmetic Sci. 2001 SepOct;52(5):265-80
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 Feughelman, M. A two-phase structure for keratin fibres. Text. Res. J. 29, 223–228 (1959).
 Popescu C & Gummer C.L., DSC of human hair: a tool for claim support or incorrect data analysis? International Journal of Cosmetic Science, 2016, 38, 433–439
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