WO2013179098A1 - Association of potassium caproyl tyrosine, l-phenylalanine and taurine and use thereof to prevent and slow down white hair - Google Patents

Description

ASSOCIATION OF POTASSIUM CAPROYL TYROSINE, L-PHENYLALANINE AND TAURINE AND USE THEREOF TO PREVENT AND SLOW DOWN WHITE HAIR

DESCRIPTION

The present invention relates to an association of potassium caproyl tyrosine, L- phenylalanine and taurine, which are capable of modulating tyrosinase expression and of suppressing free radical activity, for the purpose of acting on the physiological pigment-forming mechanism of hair bulb melanocytes and "reserve" melanocytes of the external epithelial sheath, to a composition containing such an association, and to the use thereof to prevent and slow down white hair.

Prior art

The problem of white hair has become particularly important in modern society, as evidenced by the number of compounds, formulations and treatments used to combat this problem. However, none of these treatments has been completely successful. It was therefore felt the need in the prior art to provide new means for treating and preventing white hair.

It has surprisingly been found that an association of potassium caproyl tyrosine, L- phenylalanine and taurine may provide a contribution to the solution of the problem. The present invention therefore relates to the association as claimed in independent claim 1 . Further embodiments are disclosed in the dependent claims.

Brief description of the figures

The present description is accompanied by two figures, in which:

figure 1 shows an amplification curve illustrating the quantification of changes in gene expression of tyrosinase in murine melanoma cells exposed to treatment with the association of the present invention, and

figure 2 shows tyrosinase expression after 72 hours of administration of the association according to the invention.

Summary of the invention

The present invention relates to an association (deGREY™) of potassium caproyl tyrosine, L-phenylalanine and taurine, which are capable of modulating tyrosinase expression and of suppressing free radical activity for the purpose of acting on the physiological pigment-forming mechanism of hair bulb melanocytes and of "reserve" melanocytes of the external epithelia sheath so as to prevent and slow down white hair. The association according to the invention can be administered in such a way that the three components are administered practically simultaneously, or in such a way that said active ingredients are contained in a mixture in the association.

The association can be formulated as a composition for topical cosmetic use. The use according to the present invention is in no way linked to or deducible from the use as already known.

Prior art

It is known that the amino acid tyrosine is the primary melanogenic substrate forming the basis of the biochemical schema for melanin formation. The intra- melanocyte levels of tyrosine, available for melanogenesis, are likely controlled, at least in part, by phenylalanine hydroxylase - an enzyme which converts phenylalanine into tyrosine. Tyrosinase (TYR) is an enzyme having three catalytic functions (tyrosine hydroxylase, DOPA oxidase and DHI/DHICA oxidase), likely localised at three separate sites. The most important catalytic activity is the hydroxylation of L-tyrosine to DOPA, the yield of which is negligible in the absence of tyrosinase. It is also known that pure tyrosine does not exhibit acceptable bioavailability for topical application, and is a skin irritant. Potassium caproyl tyrosine obtained by a process of tyrosine condensation with capric acid increases the bioavailability of tyrosine, providing the equivalent of approximately 10 % of pure tyrosine. The condensation product is a water-soluble lipo-amino acid, in which the capric acid, a C10 saturated fatty acid, is able to produce a hydrophilic molecule with excellent affinity for skin. In vitro tests carried out by the Irritection Assay System for determining skin irritation of ingredients have indicated that the sample should be classified as a non-irritating agent (score = 0.63). The results of the assessment regarding the effectiveness on pigmentation after UV radiation are also known. The product was compared with a placebo and an untreated control for three consecutive weeks, with use of a solar simulator. A cream containing potassium caproyl tyrosine was applied to 12 volunteers (Fitzpatrick phototypes II, III, IV) for three consecutive weeks. UV radiation was provided using a solar simulator (MUltiport Solar UV simulator Model 601 , Solar Light Company, Philadelphia, Pennsylvania, USA). The effect on skin was assessed using a colorimeter (Chromameter CR-300 minolta, Minolta GmbH, Germany) up to three weeks after application. The parameters (L^*, parameters a^*b^*, ITA value), which are sensitive to the rates of change in pigmentation intensity, were taken by colorimetric measurement of the skin before application of the product and then at various intervals. The ITA (individual typological angle) value expresses the melanin index. This is calculated as the ratio obtained by complex calculations from the parameters L^*a^*b^*. Any change to the ratio therefore could indicate a significant change in the colorimetric values. The assessment was made over three areas selected over the volunteer's back. Each area was treated using a different sample (active, placebo and untreated) with a non-occlusive patch. A fourth area of the back was exposed to UVA-UVB rays for the purpose of determining the minimum sunburn-inducing dose on unprotected skin (MEDu). The colorimetric measurements were taken on day 1 (T0) before exposure to radiation and before application. The measurements were also taken before application during the first week (T1-T3), the second week (T4-T8) and the third week (T9-T14). UVA-UVB radiation corresponding to 50 % MEDu was implemented after application of the non-occlusive patch containing the sample of the product or the placebo. The data were subject to variance analysis and to Tukey's test to determine the statistically significant differences between all the values recorded at different points over the three areas. The results showed that exogenous administration of L-tyrosine by application of potassium caproyl tyrosine in conjunction with exposure to UV rays favours endogenous activation of L- tyrosine, significantly promoting an increase in skin pigmentation.

Phenylalanine is a non-polar amino acid which is involved in the formation of the most common food proteins and which is an essential amino acid in the human organism. It can be converted into tyrosine by phenylalanine hydroxylase, which is in turn converted into L-DOPA, epinephrine and norepinephrine. It is found primarily in foods high in protein, such as meat, fish and eggs, but also in bananas, beans, nuts such as almonds and hazelnuts, in yellow pumpkin seeds and in sesame seeds.

Taurine, or 2-aminoethanesulfonic acid, is a chemical substance which is abundant in many tissues of different animals. Taurine acts primarily at neuron level, melanocyte level, and likely at keratinocyte level. Melanocytes are the cells which synthesise the pigment melanin, which is responsible for the colouring of the skin and of the appendages. The presence on the surface of human melanocytes of metabotropic glutamate receptors (activatable by glutamic acid and other agonists, including cysteine sulfonic acid) has been evidenced, the abnormal activation of which, for example in the presence of considerable amounts of cysteine sulfonic acid (CSA), causes a toxic effect at cell level: this neurotoxic effect, demonstrated previously merely in the neuron, is related to an intra-cellular increases in calcium and to the subsequent presence of an important free radical, such as nitrous oxide, which is directly involved with the mechanism for neural damage. Nitrous oxide, similarly to the superoxide anion, the hydroxyl radical and hydrogen peroxide, are important free radicals, capable of causing significant damage to cell membranes, including those of skin cells. According to the research carried out by Gerald Weissmann, it is the hydrogen peroxide produced by our body (and the main component of oxygenated water) which whitens hair, turning it grey. However, it is possible to treat grey hair at biochemical and molecular level by suppressing two decisive factors which may lead to decolouration of the hair, that is to say an increase in the free radicals produced naturally by our metabolism, such as peroxide, and a scarcity of antioxidants. Free radicals accumulate in a concentrated manner due to diet and due to the daily process of washing and drying of follicles - factors which may slowly block the production of melanin, our natural pigment. Another cause is a lack of antioxidants, which are found in reduced number in patients suffering from vitiligo, a disease which causes depigmentation of the skin. Taurine inhibits the formation of free radicals in the nerve cell. It is possible that this important anti-radical mechanism is also present in melanocytes (which, similarly to neurons, originate from the neural crest and have dendrites and receptors specific to important neuro-receptors), resulting in a significant antioxidant effect able to suppress the onset of white hair.

Potassium caproyl tyrosine, L-phenylalanine and taurine are known substances used in the pharmaceutics and cosmetics industries, although it should be noted that the activity of such individual components, used separately, is significantly inferior compared to the synergy obtained from the components with regard to their pigment-forming activity. Various preparations for topical use containing just one of potassium caproyl tyrosine, L-phenylalanine and taurine are available on the market, these preparations all being used for the promotion of skin tanning via stimulation of skin pigmentation in conjunction with exposure to UV rays. However, there is currently no product for topical use which has demonstrated the ability to modulate sufficiently tyrosine expression and to suppress free radical activity for the purpose of acting on the physiological pigment-forming mechanism of hair bulb melanocytes and of "reserve" melanocytes of the external epithelial sheath so as to prevent and slow down white hair, and above all to achieve this objective without exposure of the treated area to UV rays.

DETAILED DESCRIPTION OF THE INVENTION

Unlike known formulas, the present invention proposes an association of compounds and a composition containing said association together with tolerable additives, able to modulate sufficiently tyrosinase expression and to suppress free radical activity for the purpose of acting on the physiological pigment-forming mechanism of hair bulb melanocytes and of "reserve" melanocytes of the external epithelial sheath so as to prevent and slow down white hair, independently of exposure of the treated area to UV rays.

The process of hair greying (white hair) is, for the most part, linked to ageing. It is almost certainly the result of a reduction in melanogenesis (in the various enzyme and hormone processes which control pigment formation), and is determined by precise genetic control and by a physiological slowing down of protein synthesis. The age at which the process of white hair begins is completely specific to each individual, that is to say it is linked to the genetic "clock", which determines the onset of suspension of melanin formation. It can thus be confirmed that, on average, greying starts towards the fourth decade of life, but the range varies from extremely early cases (second decade) to late cases (sixth decade). There are no pathological implications in these individual variations in the point of onset of the physiological phenomenon of hair greying, but there are certainly partial or complete modifications to this complex mechanism for controlling melanin synthesis, as described above. In the majority of cases, the process is gradual, starting with just a few hairs and finishing with whitening of almost all hairs, but can be extremely quick, with a complete loss of colour within a few months. White hair is a phenomenon which is quite different from specific pathological conditions (albinism, piebaldism, phenylketonuria, vitiligo, etc.), which may have to be considered in very early (before the second decade) and extremely sudden cases of white hair.

The physiopathological process of hair greying: a limited number of melanocytes can produce, in a single cycle of hair growth, an amount of melanin able to pigment intensely a strand of hair measuring 1.5 metres in length. However, this significant synthesis capacity only occurs at a young age, as demonstrated by Tobin and Paus in a rather interesting article entitled "Biogerontology of the follicle/melanin unit of the hair" (2001 ).

On average, the hairs of an individual pass through 7 to 15 cycles of melanin formation during the average lifetime without grey hair of 45 years. Statistically, the average age at which white hair starts to appear is as follows

• 35 years for White and Hispanic individuals

• 39 years for Asian individuals

• 45 years for African individuals

and therefore, at 50 years, 50 % of individuals have 50 % white hair (Tobin and Paus, 2001 ).

White hair may affect individual hairs:

• with a gradual loss of pigment in more anagen cycles

• with a gradual loss of pigment in the same strand (for example during the anagen phase of a single cycle)

• when the strand of hair may grow completely depigmented

The loss of pigment in white hair is caused by a marked reduction in melanogenic activity of hair bulb melanocytes during the anagen phase. It is important to note that, at least until the sixth decade of life, the reduction in the synthesis capacity of the pigment does not coincide with a decrease in anagen activity of the hair bulb. Hair may become white, but not show any signs of reduction in hair bulb life. A study by Gao and Bedell¹ has demonstrated that there are no statistically significant changes between grey hair and naturally pigmented hair in terms of maximum central diameter and tensile strength. The only significant difference was found in the greater damage caused by UV rays to grey hair compared to pigmented hair. The proliferative capacity of keratinocytes also tends to decrease beyond the sixth decade, and primarily after the seventh. The loss of pigment in white hair may also be caused by a defect in the transfer of melanosomes to keratinocytes, although these are proximal to melanogenic melanocytes.

Residual melanocytes (evident by means of dopa oxidation reaction techniques and indicative of tyrosinase activity) remain in the piliferous bulb of white hair, but appear widened compared to normal, perhaps due to the loss of dendrites, with a lesser amount of melanosomes of smaller dimension, and other ultrastructural changes (reduction in endoplasmic organelles, in the Golgi complex, etc.). It is extremely important to note that the melanocytes in grey and white hair bulbs appear vacuolated - a common cellular response to damage caused by oxidative stress - and tend to disappear quickly. White and grey hair often demonstrate a specific difficulty in absorbing the artificial colour of a dye, presumably caused by a differentiation of the cortical keratinocytes.

RATIONALE OF THE FORMULA.

An association for topical use and a composition containing said association together with tolerable additives, said composition acting on the physiological pigment-forming mechanism of hair bulb melanocytes and of "reserve" melanocytes of the external epithelial sheath and suppressing free radical activity, were formulated.

Phenylalanine was used in the association and/or composition according to the present invention, at a titer between 99 and 101 %. Such an amino acid can be used both as L-phenylalanine and in the form of D-L-phenylalanine. The amino acid taurine is contained in the same association and/or composition at a titer between 99 and 101 %.

The composition according to the invention advantageously contains potassium caproyl tyrosine in a range between 2 and 10 % by weight, L-phenylalanine (or D-L- phenylalanine) in a range between 0.3 and 1 % by weight, and taurine in a range between 0.4 and 1.2 %, based on the total weight of the composition.

The composition according to the invention advantageously contains L- phenylalanine and potassium caproyl tyrosine in a ratio by weight of 1 :13, taurine and potassium caproyl tyrosine in a ratio by weight of 1 :8, and L-phenylalanine and taurine in a ratio by weight of 1 :1.6.

The composition according to the present invention may be formulated in the form of a foam, suspension, gel, solution or emulsion.

The present invention also relates to a process for preparing the composition, said process comprising the mixing of the components in any order and the addition of the additives.

In vitro studies have shown how the association according to the present invention formed of potassium caproyl tyrosine in synergy with L-phenylalanine and taurine reaches the deep part of the follicular ostium, modulating tyrosinase expression as early as 72 hours after application in a dose-dependent manner compared to the untreated control. The levels of expression of the enzyme in the untreated control being set to 1 , it was observed at the end of the study that the levels of expression of tyrosinase were 1.8 times higher in the cells treated with the sample containing the compound, thus suggesting a substantial increase in transcriptional activity of the tyrosinase enzyme, under the aforementioned test conditions. It is interesting to note that the action on the "follicle/melanin unit" results in an increase in the anagen time of the hair bulb and an increase in the diameter of the hair strand: in practice an improvement in the condition of the hair.

Aim of the test

The in vitro methods constitute an interesting alternative to the conventional in vivo methods for assessing the biological properties of ingredients and end products for topical or cosmetic use in accordance with current law provision, which require producers of cosmetics to verify the safety of use of the products without resorting to animal testing (Basic Council Directive N° 76/768/ EEC of 27/07/76, EC L. 262 of 27/09/1976; VI Amendment Council Directive 76/768 EEC of 14/06/1993 ECL.151 of 23/06/1993). The aim of the test was to demonstrate and quantify changes in the gene expression of tyrosinase levels in murine melanoma cells exposed to treatment with the tested substance compared to controls of the same, untreated cells.

The analysis was carried out by RT-qPCR, a very sensitive method for revealing and quantifying the mRNA coding for the protein to be analysed and therefore for activation of the corresponding gene. RT-PCR measures the fluorescence emitted by the amplicons produced during each PCR cycle.

The in vitro experimental model consists of secondary cultures of a murine melanoma called B16. It concerns cells with fibroblast like morphology, which produce melanin (source: European Collection of Cell Cultures, ECACC). For the test, the sample was dissolved in water and then diluted in the culture medium DMEM high glucose + 10% FCS, supplemented with antibiotics (penicillin 2000 Ul/ml, streptomycin 1000 Ul/ml, gentamicin 10mg/ml), at final concentrations of 0.1 mg/ml and 0.05mg/ml. The positive control aMSH was tested at 16.7 ng/ml. The cells were sown in step 17 in 6-well plates for 24 hours at a concentration of 80,000 cells per well. Fresh culture medium containing the product was then added. Untreated cells were used as a negative control, and cells treated with aMSH (human melanocytes stimulating hormone) at a concentration of 16.7ng/ml were used as a positive control. Each sample was tested twice. After 72 hours of exposure, the total RNA was purified from the cells using the reagent Trizol. The total RNA was extracted using a reagent based on guanidine thiocyanate (TRIZOL- INVITROGEN) in accordance with the instruction protocol. After precipitation and centrifugation (30' at 12,000 rpm at 4 °C), the RNA was resuspended in 20 μΙ of sterile water and its concentration was determined spectrophotometrically. 300ng of total RNA were reverse transcribed into cDNA using random primers at 37 °C for 2 hours in a thermal cycler (Applied Byosistems, Foster City, CA). The changes in the gene expression profile were analysed by real-time PCR, using SYBR green as a fluorescent molecule. Two sequences of tyrosinase primers paired in the region of the boundary between introns and exons were designed. Using the database GENEBANK, it was possible to check the uniqueness of the pairing and therefore of the gene amplification. The signal emitted by the SYBR green increased in a manner directly proportional to the amount of amplicon produced during the PCR reaction. By means of the fluorescence emitted during each cycle, it was possible to monitor the PCR reaction during the exponential phase, wherein the first significant increases in the PCR product correlate with the initial amount of template.

The graph in figure 1 shows a representative amplification curve and shows the parameters used in the quantitative analyses of the data. The intensity of fluorescence in relation to the number of cycles is shown. At the start of the PCR cycles, there is only a small change in the signal of the fluorescence, this period defining the baseline in the amplification curve. An increase in fluorescence above the baseline indicates that the PCR products are accumulating. A fluorescence value called the threshold fluorescence value is then fixed and can be found just above the baseline. The CT (cycle threshold) is defined as the number of cycles required to exceed the threshold fluorescence value (for example to exceed the base line tenfold). The CT is inversely proportional to the amount of initial target nucleic acid present in the sample. High starting levels of target nucleic acid means less time to ascertain the fluorescence levels in the PCR, and therefore low CT. The CT value is very reproducible, since the threshold value is taken during the exponential phase of the PCR, where there is a linear relation between the logarithm of the changes in fluorescence and the number of amplification cycles. Changes in the gene expression profile are measured using the CT comparative method (AACT method). The data are normalized with respect to some control genes, such as 28S ribosomal RNA, actin or cyclophilin. Briefly, the AACT method enables a relative quantization to the template in the form of expression levels relative to the normalizer. It is expected that the normalizer will have expression levels higher than those of the target (therefore a low CT value). For quantitative analysis, the difference between the CT value of the target and that of the normalizer (ACT) is first established.

ACT = CT (target) - CT (normalizer)

This value is calculated for each sample which has to be evaluated from a quantitative point of view. The untreated sample will be used as a reference value for all the comparisons which will be made. The AACT method then calculates the difference between each ACT and that of its control. If the value of the control represents the minimum level of expression, an increase in the expression levels of the target gene, and therefore negative AACT values, would be expected under the test conditions (because the ACT of the control will be lower than that of the samples). By contrast, if a decrease in the expression levels of the target gene is expected under the test conditions, the AACT will have positive values (because the ACT of the control will be greater than that of the samples).

The last phase in the quantization calculation is the conversion of these values according to the following formula:

Fold change = 2 - AACt

Results and conclusions:

The sample is able to slightly modulate tyrosinase expression after 72 hours of treatment (as can be seen in figure 2) in a dose-dependent manner, with maximum effect at a concentration of 0.1 mg/ml. In particular, it can be seen that, with respect to the untreated control (CN), in which the enzyme expression levels are set to 1 , in the cells treated with the sample, the tyrosinase expression levels are 1.8 times higher, thus suggesting an increase in tyrosinase enzyme transcription activity, under the aforementioned test conditions.

Bibliography

1 . Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y) 1992;10(4):413-7.

2. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y) 1993; 1 1 (9): 1026- 30).

3. Lee LG, Connell CR, Bloch W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res 1993;21 (16):3761-6.

4. Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 1995;4(6):357-62.

5. A.D. Katsambas and A.J. Stratigos, Depigmenting and bleaching agents: coping with hyperpigmentation. Clin Dermatol (19)4:483-488,2001

6. E.V. Curto, C.Kwong, H. Hermersdorfer, H. Glatt, C. Santis, V. Virador, V.J. Hearing Jr and T.P. Dooley, Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors. Biochem Pahrmacol, 57:663-672, 1999.

Based on the evidence, it has been demonstrated that the association and/or composition according to the invention (containing potassium caproyl tyrosine and L-phenylalanine and taurine) is able to modulate tyrosinase expression, and its effect has been tested with regard to the doses and formulations below.

Exemplary compositions according to the inventions are listed below:

Example 1 Anti-grey lotion

Aqua 45.7 % - Alcohol 35 % - Propylene Glycol 5 % - Panthenol/Glycerol 3 % - Potassium Caproyl Tyrosine™ 6.5 % - Taurine™ 0.8 % - L- Phenylalanine™ 0.5 % (deGREY™) - Saccharomyces/Silicon Ferment, Saccharomyces/Magnesium ferment, Saccharomyces/Copper, Ferment, Saccharomyces/lron Ferment, Saccharomyces/Zinc Ferment 1 %.

Example 2 Anti-grey restorative mask

Aqua 79.45 % - Potassium Sorbate 0.25 % - Phenoxyethanol 0.6 % - Hydroxypropyl Starch Phoshate 6 % - Cetyl Alcohol 3 % - Behent monium Chloride 2.5 % - Hydroxypropyltrimonium Hydrolyzed Corn Starch 1 % - Dimethicone PEG-8 Meadowfoamate 0.5 % - Sodium Benzoate 0.3 % - Panthenol 1 .5 % - Cetrimonium Chloride 1 % - Tocopheryl Acetate 0.3 % - Parfum 0.5 % - Sodium Benzoate 0.3 % - Potassium Caproyl Tyrosine™ 2 % - Taurine™ 0.5 % - L-Phenylalanine™ 0.3 % (deGREY™)

Example 3 Anti-grey shampoo

Sodium Lauryl Sulfoacetate/Disodium Laureth Sulfosuccinate/Aqua 10% - Sodium Lauroyl Sarcosinate 8% - Coco-Glucoside 4 % - Sodium Cocoyl Glutamate 5 % - Aqua, Cocamidopropyl Betaine 15% - Dimethicone PEG-8 Meadowfoamate 1 % - Panthenyl Hydroxypropyl/Steardomonium Chloride 1 % - Silicone Quaternium-22 1 % - Potassium Caproyl Tyrosine™ 6.5 % - Taurine™ 0.5 % - L-Phenylalanine™ 0.3 % (deGREY™) - Saccharomyces/Silicon Ferment, Saccharomyces/Magnesium Ferment, Saccharomyces/Copper Ferment, Saccharomyces/lron Ferment, Saccharomyces/Zinc Ferment 1 % - Phenoxyethanol 0.6% - Sodium Benzoate 0.3% - Potassium Sorbate 0.3 % - Aqua 43.9 % - Sodium Chloride 0.2 % - Polyquaternium-10 0.2% - Parfum

Claims

1 . An association comprising potassium caproyi tyrosine, L-phenylalanine and taurine.

2. The association according to claim 1 , comprising D-L-phenylalanine instead of L-phenylalanine.

3. A composition comprising the association according to claim 1 or 2, together with tolerable additives.

4. The composition according to claim 3, characterised in that the potassium caproyi tyrosine is contained in a range between 2 and 10 % by weight, L-phenylalanine is contained in a range between 0.3 and 1 %, and taurine is contained in a range between 0.4 and 1 .2 %, based on the total weight of the composition.

5. The composition according to at least one of claims 3 or 4, characterised in that L-phenylalanine and potassium caproyi tyrosine are contained in a ratio by weight of 1 : 13.

6. The composition according to at least one of claims 3 to 5, characterised in that taurine and potassium caproyi tyrosine are contained in a ratio by weight of 1 :8.

7. The composition according to at least one of claims 3 to 6, characterised in that L-phenylalanine and taurine are contained in a ratio by weight of 1 :1.6.

8. The composition according to any one of the preceding claims, wherein the composition is in the form of a foam, suspension, gel, solution or emulsion.

9. A process for preparing a composition according to at least one of claims 3 to 8, said process comprising the mixing of the components in any order and the addition of the additives.

10. Use of the composition according to any one of claims 3 to 8 as a cosmetic in the prevention and treatment of white hair.