Influence of Oxidative and/or Reductive Treatment on Human Hair (I): Analysis of Hair-Damage after Oxidative and/or Reductive Treatment

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1 JOURNAL OF OLEO SCIENCE Copyright 2003 by Japan Oil Chemists Society JOS Influence of Oxidative and/or Reductive Treatment on Human Hair (I): Analysis of Hair-Damage after Oxidative and/or Reductive Treatment Koji TAKADA, Akira NAKAMURA, Naoko MATSUO, Akira INOUE, Keita SOMEYA and Hisao SHIMOGAKI Biological Science Research Center, Lion Corporation (Tajima 100, Odawara, Kanagawa , JAPAN) Edited by T. Itoh, Kitasato Univ., and accepted May 27, 2003 (received for review March 5, 2003) Abstract: We devised a damaged hair model to confirm hair-damage was caused not only by chemical stress but also by physical and environmental stress. We observed morphological changes (e.g. decreasing cuticle layers, scale lift, hair swelling) in the hair model and analyzed alterations in the hair composition (e.g. cysteic acid, protein elution, melanin granule). We devised oxidative and reductive treatments (e.g. perming, bleaching) as the chemical stresses. We also devised a system for testing physical and environmental stresses. This consisted of ultraviolet ray (UV) irradiation, brushing, treatment with detergent, drying and heating. These factors are believed to cause hair-damage during normal everyday life. Scale lift was observed and this phenomenon was propagated depending on the amount of physical and environmental stress. The amount of exfoliated cuticles and cysteic acid in the cuticles also tended to increase with the progress of the physical and environmental treatments. The tendency was more remarkable with strong chemical treatments. The swelling of the hair, protein elution and the amount of solubilized melanin granule exhibited similar trends. These trends, though, were not as remarkable as those observed in the cuticle layers. Using the damaged hair model that included physical and environmental stresses, we demonstrated that stresses from everyday life, as well as chemical stresses, effected hair damage. These influences were enhanced depending on the degree of chemical treatment. This tendency was more remarkable in cuticle layers than in the cortex and medulla parts. Key words: damaged hair, chemical stress, physical and environmental stress, properties of hair components, morphological hair property 1 Introduction Hair is subjected to a number of natural and artificial conditions. These include chemical stress (e.g. perming, dyeing, bleaching), physical stress (e.g. brushing, heating), and environmental stress (e.g. UV irradiation, pollution) (1,2). Prior studies have reported that these stresses disrupt the mechanical and morphological properties of hair, such as decreasing the cuticle layers, scale lift, and split ends, and lead to hair damage (3-6). Fashion dictates that people want to maintain their hair in a natural and beautiful condition whether or not it has been treated by perming, dyeing, or bleaching. We consider hair-damage can occur due to two factors that exist in modern life. Primary damage is chemical stress, such as perming or dyeing, and these treatments are considered to cause strong damage to hair. The second factor is physical or environmental stress in every- Correspondence to: Keita SOMEYA, Biological Science Research Center, Lion Corporation, Tajima 100, Odawara, Kanagawa , JAPAN keisliof@lion.co.jp Journal of Oleo Science ISSN print / ISSN online 541

2 K. Takada, A. Nakamura, N. Matsuo et al. day life, such as brushing or sunlight-exposure, and these stresses are considered to be milder than the primary damage. Such kinds of damage, though, accumulate. Previous papers have reported that chemical stress leads not only to morphological changes but also to alterations in the biological composition of the hair, such as amino acids or lipid composition (7-9). Concerning permed hair, Kon et al. observed a significant decrease in microfibril protein and an increase in high molecular-weight protein on the tip end (10). In addition, Benard et al. reported an increase in induced chemiluminescence (ICL-H) in permed and colored hair (11). However, there has been little research into analyzing the changes in biological composition for secondary hair-damage factors. In this study, we observed morphological changes (e.g. decreasing cuticle layers, scale lift, hair swelling) and analyzed the alterations in the composition (e.g. cysteic acid, protein elution, melanin granule) after primary and secondary hair damage. 2 Experimental 2 1 Hair Samples Hair fibers used in this study were chemically untreated samples taken from a Japanese woman. The hairs were divided into approximately 0.2 g, 11 cm lengths for each tress, and secured at the non-tip end. These tresses were washed with 3 % sodium dodecyl polyoxyethylene sulfate (Sannol MT-1430, Lion Co., Tokyo, Japan) for 30 s and rinsed three times in 2000 ml of water at 40 for 1 min. They were dried at 55 for 10 min and then brushed to disentangle the knots. 2 3 Preparation of Artificially Damaged Hairs Chemical treatment is shown in Scheme 1 and the physical and environmental treatment is shown in Scheme 2. Physical and environmental treatment are postulated to cause hair-damage in everyday life, such as ultraviolet ray (UV) irradiation, brushing, shampooing, drying and styling. One cycle of treatment corresponded to usual daily behavior. 2 4 Scanning Electron Microscopy (SEM) SEM observation was carried out following the conventional method. Hair fibers were selected at random, sputtered with gold, and examined with a S2380 scanning electron microscope (Hitachi Co., Tokyo, Japan). 2 5 Hair Swelling Test Hair samples were immersed in water for 5 min. The diameter of the hair was measured using a digital microscope VH-8000 (Keyence Co., Osaka, Japan) both before and after immersion. We defined the ratio of the diameter increase of before to after immersion as the degree of hair swelling. 2 6 Analysis of Cysteic Acid Content Cysteic acid content was determined by amino acid analysis using AccQ-Tag (Milford, MA, USA). Ten milligrams of hair fibers were hydrolyzed with gaseous hydrochloric acid (HCl) at 110 for 21 h. After removing the excess HCl in vacuo, the amino acids were resolubilized with 500 ml of 20 mm HCl, and 1 ml of hydrolysate was collected. Amino acids in the hydrolysate were labeled with 6-aminoquinolyl-N- 2 2 Chemicals Chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan) except where otherwise detailed. Scheme 1 Flow Scheme for Chemical Treatments. 542

3 Influence of Oxidative and/or Reductive Treatment on Human Hair (I) hydroxysuccinimidyl carbamate (Waters Co., Milford, MA, USA) and analyzed by reversed phase HPLC according to the AccQ-Tag method (12). 2 7 Protein Elution Test Twenty five milligrams of hair fibers were immersed in 1 ml of 40 mm sodium phosphate buffer (ph 7.0) at 37 for 24 h. The extracts were dried and hydrolyzed with gaseous HCl at 110 for 21 h. Amino groups in the hydrolysate were labeled with fluorescamine (FLUKA, Buchs, Switzerland) according to Ueno et al. (13) and fluorescence from the sample was measured. The amount of protein was calculated using the calibration curve of glycine. 2 8 Measurement of Solubilized Melanin The melanin content in hair was measured after digesting with proteolytic enzyme. Twenty mg of hair fibers were incubated in 1 ml of 40 mm borate buffer (ph 9.3) containing 100 mm Dithiothreitol (DTT) and 30 Novo protease units (NPU)/mL Esperase (Novozymes Japan Ltd., Chiba, Japan) at 37 for 18 h (14). The suspension that was obtained was diluted with 20 times of the amount of water. Furthermore, HCl was added to the suspension, which included the degraded hair, to make up a final concentration of 1 M. Supernatant was obtained by centrifuging, and optical density (OD) at 400 nm was measured as the concentration of solubilized melanin. 2 9 Cuticle Removal Test We conducted a cuticle removal test according to Kon et al. (6) with a slight modification. Hair fibers of weight 50 mg were cut into 20 mm lengths. The hairs were placed in 2.5 ml of 1% sodium dodecyl polyoxyethylene sulfate in 10 mm sodium phosphate buffer (ph 7.0) and sonicated using an ultrasonic cleaner B2200 (Branson Cleaning Equipment Co, Shelton, WA, USA) at 40 for 10 min. The hair was washed 3 times in water. The water and sonicated fluid were then centrifuged at 12,000 g for 15 min. The supernatant was discarded and the pellet was resuspended in 1.5 ml of water. The suspension was transferred to a 2mL micro tube, then centrifuged again. The precipitate was washed with ethanol and the pellet was dried in vacuo and weighed Sensory Assessment Texture and appearance in the damaged hair were sensorially evaluated by a couple of specialists. The parameter tested for texture was the sense of smoothness under combing, and for appearance was the degree of twining around before combing and the broadness of the tip end after combing. Scheme 2 Flow Scheme for Physical and Environmental Treatment. One cycle of treatment was postulated as the usual behavior in a day. 543

4 K. Takada, A. Nakamura, N. Matsuo et al. 3 Results 3 1 Hair Morphology and Sensory Properties Figure 1 is a SEM image of the hair surface. Disorder in the cuticle edge was almost not present on the hair surface immediately after the bleaching. Scale lift was observed on the hair surface after the physical and environmental treatment, and the scale lift was more severe where it had been treated 14 times than where it had been treated 7 times. However, apparent scale lift was not observed on the hair that was treated 28 times. Similar surface changes were also observed in the hair that was subjected to a different chemical treatment (bleaching for 60 min, perming and bleaching treatment, data not shown). Furthermore, the sense of smoothness when brushing decreased and the broadness of the tip end after combing increased where it had been treated over 14 times. On the other hand, twining in the hair tress was observed only for cases between 14 to 21 times. 3 2 Cuticle Removal Test Figure 2 shows the amount of the cuticle exfoliated from the hair surface due to ultrasonics. The degree of cuticle removal tended to increase depending on the strength of chemical treatment. The amount of exfoliated cuticles also tended to increase with the increase in physical and environmental treatment. This tendency was more remarkable with severe chemical treatments. 3 3 Cysteic Acid Content of the Cuticle Figure 3 shows the cysteic acid content of the cuticle that was obtained in the cuticle removal test. The trend of the results was similar to that in Fig. 2. Thus, not only the strength of chemical treatment, but also the amount of physical and environmental treatment led to an increase in cysteic acid in the cuticles. 3 4 Hair Swelling Test The degree of hair swelling after soaking is shown in Fig. 4. Hair diameter after the chemical treatment increased and the tendency depended on the strength of the chemical treatments. A similar tendency was obtained after the physical and environmental treatment. However, the observation was not as remarkable compared to the observations made during cuticle removal or cysteic acid analysis (Fig. 2,3). 3 5 Protein Elution Test Figure 5 shows the amount of protein eluted from hair after each process. The trend of the result was similar to that in Fig. 4. Thus, changes of protein elution due to the chemical treatment were remarkable but changes due to physical and environmental treatment were not so apparent. 3 6 Changes in the Amount of Solubilized Melanin Changes in solubilized melanin after treatment are shown in Fig. 6. We measured the OD at 400 nm to calculate the amount of melanin. After physical and envi- Fig. 1 Photomicrographs of the Damaged Hair Surface. All hair samples were bleached for 30 min, as described in Scheme 1. After bleaching the hair samples were treated with physical and environmental stresses (PES), as described in Scheme 2. The hair surface was observed using SEM after all the treatment was completed. A, only bleaching; B, treated 7 times with PES after bleaching; C, treated 14 times with PES after bleaching; D, treated 28 times with PES after bleaching. Scale bar is 25 micro meters. 544

5 Influence of Oxidative and/or Reductive Treatment on Human Hair (I) Fig. 2 Exfoliated Cuticles after a Combination of Chemical Stresses and Physical and Environmental Stresses. The processes for the chemical stresses (CS) and physical and environmental stresses (PES) are described in Scheme 1 and Scheme 2. After treatment, the exfoliated cuticles were measured as described in the experimental section. UT, untreated hair; B30, bleached for 30 min; B60, bleached for 60 min; PB30, bleached for 30 min after perming;, no PES treatment;, seven times PES treatment;, fourteen times PES treatment;, twenty eight times PES treatment. Fig. 4 Degree of Hair Swelling after Hair Stress Treatment. The treatment was the same as in Fig. 2. After treatment, the degree of hair swelling was measured as described in the experimental section. All abbreviations and symbols are the same as in Fig. 2. Fig. 5 Amount of Extracted Protein after Hair Stress Treatment. The treatment was the same as in Fig. 2. After treatment, the amount of extracted protein was measured as described in the experimental section. All abbreviations and symbols are the same as in Fig. 2. Fig. 3 Cysteic Acid in Cuticles after Hair Stress Treatment. The treatment was the same as in Fig. 2. After treatment, cysteic acid in the cuticles was measured as described in the experimental section. All abbreviations and symbols are the same as in Fig. 2. ronmental treatment, solubilized melanin tended to be greater than before the treatment and the order depended on the number of times processed. The tendency, though, was not so remarkable. 4 Discussion We devised two different damaged hair models for the present study. One was a chemical stress (primary stress) model in which hair was treated by perming or bleaching. The other was a physical and environmental stress (secondary stress) model where hair was subjected to damage that occurs in everyday life, for example UV exposure, brushing, shampooing, drying and styling. Using a combination of these models we evaluated changes in the morphological properties of the hair (e.g. cuticle exfoliation, scale lift, and hair swelling) 545

6 K. Takada, A. Nakamura, N. Matsuo et al. Fig. 6 Amount of Solubilized Melanin after Hair Stress Treatment. The treatment was the same as in Fig. 2. After treatment, the amount of solubilized melanin was measured as described in the experimental section. All abbreviations and symbols are the same as in Fig. 2. and alterations of the composition in the hair (e.g. cysteic acid, protein elution and melanin granule). We revealed that secondary stresses were also significant factors for causing hair damage. After the physical and environmental treatment, scale lift occurred and this led to a decline in the luster of hair (15). As shown in Fig. 1, we observed scale lift on the hair surface with a combination of primary and secondary damages. In addition, Swift proposed the hypothesis that physical force when washing and combing hair causes fractures of the cuticle (16). We also confirmed exfoliation of cuticles after repeated physical and environmental treatment (Fig. 1D,2). On the other hand, there was almost no morphological change immediately after the chemical treatment process (Fig. 1A). However, there have been many reports suggesting chemical treatment causes changes in the hair composition, for example an increase in cysteic acid that is derived from oxidation of the disulfide bond (9,10) and protein elution (17). Changes in these components could be preparatory factors for causing morphological changes after physical and environmental stress. The amount of exfoliated cuticles increased and that correlated with the intensity of the chemical treatment (Fig. 2). Disulfide cross-link, which is included in cystine, and isopeptide were reported as protein cross-links in cuticles, with the former a major cross-link (18). It was also reported that cysteic acid is derived from cystine by oxidative cleavage in hair fiber (10). The cuticles contained approximately 18 % cysteine, which was reduced from cystine (19), and large amounts of cysteic acid were generated in exfoliated cuticles. After the physical and environmental treatment, the cysteic acid content was much higher than after only chemical treatment (Fig. 3). Therefore, the destruction of the crosslink probably decreased the strength of the cuticle and this decrease would cause scale lift and exfoliation of the cuticles during the hair-damage. It was shown in Fig. 3 that cysteic acid increased in cuticles depending on the number of physical and environmental treatments used. Benard et al. reported that peroxide remained in hair after chemical treatment (e.g. bleaching) and UV irradiation (11). We reported separately that the peroxide propagated after a combination of primary and secondary damage, and that antioxidants were effective against hair-damage (20). Therefore, it can be concluded that repetition of secondary treatment after primary damage causes a propagation of peroxide and the destruction of the disulfide cross-link in hair. In the cuticle removal test, the amount of exfoliated cuticles from permed and bleached hair was remarkably higher than that from only bleached hair. It is therefore likely that exfoliation is not only due to the destruction of the disulfide cross-link but also to other factors, for example protein elution (21,22) and labile protein formation (23) that was probably reduced during the perm treatment. The changes in the tactile properties after the physical and environmental damage seems to be caused by the cuticle changes that were described above. Scale lift formation and twining were observed at the same intensity of secondary treatment, therefore both phenomena are probably related to each other. In addition, it can be concluded that the smoothness and broadness of the tip end are also related to the property of hair fiber, for example the friction of the hair surface or hydrophobicity of the hair, which is correlated with hair surface lipid (24). Further examination needs to be done to reveal the relation of these phenomena in detail. The degree of hair swelling was slightly increased by further physical and environmental damage. Hair swelling may be related to the oxidative distraction of the disulfide cross-link in hair fibers. However, we could detect the increase of cysteic acid, which was derived from oxidative cleavage of disulfide cross-link, not in the hair fiber but in the cuticle (Fig. 3). Therefore, we consider that the phenomenon is probably related to cavitation. An increase in the amount of pro- 546

7 Influence of Oxidative and/or Reductive Treatment on Human Hair (I) tein elution and solubilized melanin content (Fig. 5, 6), possibly caused by the remaining peroxide after hairdamage, could relate to the formation of cavities. We can conclude that not only primary damage but also secondary damage, which is caused by everyday behavior, influences morphological changes and alters the hair components. In order to prevent oxidative and/or reductive damage to hair, it is important to clarify the detailed correlation between these hair stresses and the changes of hair properties, and how an antioxidant could be effective against this hair damage. Recently, we found that hydrophilic extracts from Rosmarinus officinalis L. (HER) had an excellent antioxidant effect on the hydrophilic status. HER also exhibited preventive efficacy on damaged hair where the surface is more hydrophilic than that of usual hair. We will report separately on the effect of HER on hairdamage in detail (20). Acknowledgement We thank Ms. Yukako Mizushima for her advice in devising the procedure for damaged model treatments. We are also grateful to Ms. Midori Morishima for her technical assistance in the treatment and analysis of hair. References 1. M.L. TATE, Y.K. KAMATH, S.B. RUETSCH and H.D. WEIGMANN, Quantification and Prevention of Hair Damage, J. Soc. Cosmet. Chem., Vol. 44, (1993). 2. S.B. RUETSCH, Y. KAMATH and H.D. WEIGMANN, Photodegradation of Human Hair: An SEM Study, J. Cosmet. Sci., Vol. 51, (2000). 3. C.M. PANDE and J. JACHOWICZ, Hair Photodamage Measurement and Prevention, J. Soc. Cosmet. Chem., Vol. 44, (1993). 4. S.E. KELLY and V.N.E. ROBBINSON, The Effect of Grooming on the Hair Cuticle, J. Soc. Cosmet. Chem., Vol. 33, (1982). 5. J.A. SWIFT and A.C. BROWN, The Critical Determination of Fine Changes in the Surface Architecture of Human Hair due to Cosmetic Treatment, J. Soc. Cosmet. Chem., Vol. 23, (1972). 6. R. KON, A. NAKAMURA and K. TAKEUCHI, Artificially Damaged Hairs: Preparation and Application for the Study of Preventive Ingredients, Int. J. Cosmet. Sci., Vol. 20, (1998). 7. H. ZAHN and H.G. GATTNER, Hair Sulfur Amino Acid Analysis, in Formation and Structure of Human Hair (P. JOLLES, H. ZAHN and H. HOCKER, eds.), Birkhauser, Basel, pp (1997). 8. S.H. BONG and H. ZAHN, Contributions to the Chemistry of Human Hair: II. Lipid Chemical Aspects of Permanently Waved Hair, Int. J. Cosmet. Sci., Vol. 11, (1989). 9. J. CHAO, A.E. NEWSOM, I.M. WAINWRITE and R.A. METHEWS, Comparison of the Effects of Some Reactive Chemicals on The Proteins of Whole Hair, Cuticle and Cortex, J. Soc. Cosmet. Chem., Vol. 30, (1979). 10. R. KON, A. NAKAMURA, N. HIRABAYASHI and K. TAKEUCHI, Analysis of the Damaged Components of Permed Hair Using Biochemical Technique, J. Cosmet. Sci., Vol. 49, (1998). 11. S. BENARD, H. NERENZ, M. ROHR and K. SCHRADER, ICL-H (Induced Chemiluminescence of Human Hair) a New Method for Quantitative Analysis of Hair-stress, IFSCC Magazine, Vol. 4, (2001). 12. S.A. COHEN and D.P. MICHAUD, Synthesis of a Fluorescent Derivatizing Reagent, 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate, and its Application for the Analysis of Hydrolysate Amino Acids via High-performance Liquid Chromatography, Anal. Biochem., Vol. 211, (1993). 13. R. UENO, K. SAKANAKA and Y. HORIGUCHI, Purification of a Pepstatin Insensitive Protease from Mackerel White Muscle, Nippon Suisan Gakkaishi, Vol. 54, (1988). 14. A. NAKAMURA, R. KON and K. TAKEUCHI, Japan Kokai, C.R. ROBBINS, Physical and Cosmetic Behavior of Hair, in Chemical and Physical Behavior of Human Hair Fourth Edition, Springer Verlag, New York, p. 276 (2002). 16. J.A. SWIFT, Fine Details on the Surface of Human Hair, Int. J. Cosmet. Sci., Vol. 13, (1991). 17. A.S. SANDHU, R. RAMACHANDRAN and C.R. ROBBINS, A Simple and Sensitive Method using Protein Loss Measurements to Evaluate Damage to Human Hair during Combing, J. Soc. Cosmet. Chem., Vol. 46, (1995). 18. H. ZAHN, Wool is not Keratin Only, in Proc. 6th Int. Wool Textile Res. Conf., Pretoria, pp (1980). 19. J.A. SWIFT and B. BEWS, The Chemistry of Human Hair Cuticle-III: The Isolation and Amino Acid Analysis of Various Subfractions of the Cuticle Obtained by Pronase and Trypsin Digestion, J. Soc. Cosmet. Chem., Vol. 27, (1976). 20. K. TAKADA, K. SOMEYA, N. MATSUO, Y. NISHIDA and T. HAYASHI, Influence of Oxidative and/or Reductive Treatment on Human Hair (II): Effect of Hydrophilic Extracts from Rosmarinus officinalis L. on Oxidative and/or Reductive Hair-damage, J. Oleo Sci., Vol. 52, (2003). 21. H. TSUSHIMA, A. UEKI, H. SUMI and V.K. HPOSU-HAVU, Purification and Characterization of a Cystatin-type Cysteine Proteinase Inhibitor in the Human Hair Shaft, Arch. Dermatol. Res., Vol. 284, (1992). 22. K. KIZAWA, H. UCHIWA and U. MURAKAMI, Highlyexpressed S100A3, a Calcium-binding Protein, in Human Hair 547

8 K. Takada, A. Nakamura, N. Matsuo et al. Cuticle, Biochim. Biophys. Acta, Vol. 1312, (1996). 23. T. INOUE and K. KIZAWA, Labile Proteins Accumulated in Damage Hair upon Permanent Waving and Bleaching Treatments, J. Cosmet. Sci., Vol. 53, (2002). 24. L.N. JONES and D.E. RIVETT, The Role of 18-Methyleicosanoic Acid in the Structure and Formation of Mammalian Hair Fibres, Micron, Vol. 28, (1997). 548