INTRODUCTION

Ageing is a complex process involving various genetic, hormonal, and environmental mechanisms. Similar to skin, the scalp and hair are subject to internal (chronological) ageing and to external ageing due to environmental factors (1). Proteins are the building blocks for individual hair fibre (making up 65 to 95% of the hair). These are structurally held together by chemical hair bonds that ensure structural integrity of hair fibre and its strength. The hair bonds are all affected by ageing:

• Disulphide bonds are strong permanent chemical bonds that form between sulphur atoms in the amino acid cysteine, creating a cross-link between keratin molecules in the hair. These bonds provide hair with strength and elasticity. With age, these bonds weaken due to decreased cysteine production and are further compromised by chemical treatments, UV radiation and pollution (2).
• Hydrogen bonds are weaker, temporary physical bonds that form between water molecules and proteins in hair. These bonds contribute to the hair’s flexibility and are easily broken by water and heat. With age, the hair’s moisture content diminishes, reducing the number of hydrogen bonds. External factors like frequent use of heat styling tools, excessive washing and environmental dryness exacerbate this reduction (3).
• Ionic (or salt) bonds are temporary bonds formed between acidic and basic amino acids in the hair. These bonds are sensitive to pH changes and can be disrupted by acidic or alkaline substances. As the scalp ages, its natural pH balance varies, weakening the ionic bonds in the hair. External factors such as harsh shampoos and environmental pollutants can also disrupt these bonds (4).

As a consequence of ageing, hair deteriorates on several levels:

• Molecular: hair becomes drier, more fragile and brittle, with loss of colour.
• Structural: hair becomes thinner and experiences changes in texture.
• Visual: hair loses strength and volume, becoming more prone to breakage.

In this paper, Oat Peptide Powder (OPP) (Avena sativa (oat) peptide) is obtained from an oat by-product following an enzymatic extraction, filtration and purification step to target and isolate a novel oat peptide product. In this context, the objective of these studies was to investigate the ingredient’s effect on preventing hair ageing and repairing damage. Ex vivo studies were performed to evaluate OPP ability to prevent keratins deterioration, restructure hair fibre and strengthen hair.

MATERIALS AND METHODS

Peptides Identification
Peptide purity: The material was digested with concentrated sulphuric acid using copper-titanium as a catalyst to convert organic nitrogen to ammonium ions. The resulting digest was made alkaline, distilled into excess boric acid and the ammonia trapped by the boric acid is titrated with standard hydrochloric acid. The nitrogen content was calculated from the amount of ammonia produced; standard factors are then used to convert total nitrogen to protein equivalent.

Peptides molecular weight: The molecular weight of the peptides was determined using liquid chromatography-mass spectrometry (LC-MS). OPP was dissolved in 2.5% sodium deoxycholate. The solution was treated with dithiothreitol and iodoacetamide to alkylate cysteine residues, desalted using C18 and then directly analysed by LC-MS. After alkylation, an aliquot was digested with trypsin and analyse by MS using the same procedure. Both data sets (+/- trypsin) were processed in the Proteome Discoverer software (PD 2.4.1, Thermo).

Amino acid distribution: The material was hydrolysed in aqueous hydrochloric acid to break peptide bonds, then pH was adjusted, brought to volume with a loading buffer and filtered. Amino acids were separated using an amino acid analyser and the detection was carried out using post-column derivatisation with a ninhydrin reagent at 440 and 570 nm.

Reparation of hair keratins (molecular reparation)
An ex vivo study was performed using 3 straight, healthy, hair tresses for each condition:

Table 1. Summary of the experimental conditions (Leave on application).

Evaluation of oxidation damage: Hair shafts were cryo-preserved, snap-frozen in liquid nitrogen and cross-sectioned. Slices of 5 μm were obtained using a cryostat (Leica). Carbonyls were labelled in situ using a fluorescent probe (Ex=647 nm / Em=650 nm) functionalised to specifically bind carbonyl groups (5).
Qualitative Data: Fluorescent images were collected with an epi-fluorescent Microscope (ThermoFisher, Evos M5000) and analysed with ImageJ software. Identical conditions of acquisition (40x objective) were used for all samples.
Quantitative Data: The intensity of carbonylation was obtained by the integration of the specific fluorescence signal normalised by the evaluated area. Three images per condition were used to quantify the carbonylation levels; the mean value and standard deviation were obtained per condition (on the whole section) and per compartment (cortex and cuticle).

Restructuration of hair fibre (structural reparation)
An ex vivo study was performed using 3 straight, healthy, hair tresses for each condition:

Table 2. Summary of the experimental conditions (Leave on application).

Evaluation of hair fibre: Hair segments were transferred on slides following KAMAX’s protocol (6). The XPolar compatible slides were analysed using a KProbe slide scanner. For each condition, 30 independent hair segments of 1 cm length were analysed. XPolar technology is based on an imaging approach to measure the birefringence. The propagation of the light that goes through the hair segment is different whether the polarisation of the light is parallel or not to the keratin fibre. The observed difference results in a delay of light propagation that is called birefringence. The determination of the keratin birefringence of each hair segment by K index (depending on hair thickness) was performed and analysed using GraphPad Prism 9 software.

Strengthening of hair (visual reparation)
An ex vivo study was performed using 3 straight, healthy, hair tresses for each conditioner: placebo, 1% Oat Peptide Liquid and 1% OPP conditioners. For each conditioner, the treatment was the same:

1. Application of standard SLS shampoo.
2. Determination of initial hair strength (T0) by Tensile Test using a Universal test machine (measurement of the force per unit area, or tensile stress, required to break the hair, which characterises the structural integrity of individual hair fibres).
3. Thermal aggression: 8 applications of hair straighteners (> 230°C) to damage the hair surface.
4. Application of test conditioner: Soaking hair in water at 37°C for 1 minute, applying 2 g of conditioner and leaving for 4 minutes, rinsing with water at 37°C and then drying the tress naturally.
5. Determination of final hair strength (TF).

RESULTS AND DISCUSSION

Peptides Identification
Peptide purity: OPP contains 90 % peptides.

Peptides molecular weight (MW): The MW of peptides present in OPP is directly related to their function at the hair level. Peptides are present in, predominately the medium (3,000 – 1,000 Da) and low (<1,000 Da) range, to maximise hair efficacy. Medium MW can form ionic or hydrogen bonds with the hair and have a repairing role of the cuticle (7,8). Low MW allow peptides to penetrate and perform a repairing action of the hair fibres from the inside (7,8). OPP peptide distribution is ideal for hair care, for acting both internally and externally.

Amino acid distribution: OPP has a high amino acid content with high substantivity for keratin (Fig. 1). Keratin can be linked to the following amino acids:

• Acidic amino acids through ionic bonds.
• Polar amino acids which establish hydrogen bonds.
• Sulphuric amino acids which are linked by disulphide bonds.

Conclusion

Aged hair often exhibits characteristics such as dullness, brittleness, fragility and a fine texture. Oat Peptide Powder is designed to specifically target the needs of fragile and ageing hair. By efficiently strengthening and addressing all three types of hair bonds, Oat Peptide Powder offers comprehensive repair and immediate perceivable benefits.
Through its molecular level repair of keratins, structural repair of hair keratin fibre organisation and density, and visual strengthening of hair, Oat Peptide Powder application results in healthier, more structured and repaired hair strands, combatting the visible signs of hair ageing.