皮肤-头发的近红外分析

CONTRIBUTED REVIEW 4. Infrared and Raman Studies of Skin and Hair: A review of cosmetic spectroscopy Kathleen Martin Unilever HPC USA 3100 Golf Road Rolling Meadows, IL 60008 USA Tel: (847) 734-3714 Fax: (847) 734-3686 email: kathleen.martin@unilever.com Introduction Each year the cosmetic industry reaps billions of dollars from sales of products designed to enhance skin and hair. Products to cleanse, moisturize or condition skin and hair abound. In order to compete for the consumer’s dollar, the industry has expended much effort on improving our understanding of the composition and structure of skin and hair and on the nature of the interactions between these substrates and the products themselves. Vibrational spectroscopy, both in vitro and in vivo, has been an important tool in these studies. Presented here is an overview of infrared, Raman and near-infrared spectroscopic studies of skin and hair in the cosmetic industry. 1. Composition and Structure of Skin and Hair Skin and hair both function as protective barriers between the body and the environment. The outermost layer of skin, the stratum corneum (SC), is of particular interest to cosmetic scientists as this is the layer affected by cosmetic products. In addition to defending the body against chemical and bacterial attack, the SC regulates evaporative water loss through the skin. Scalp hair plays a similar role by acting as a cushion for the head, protecting the scalp from sunlight, and helping to maintain body heat. Both skin and hair are composed primarily of keratin, a tough, insoluble protein also found in nails, hooves, horns and feathers, plus smaller amounts of lipids and water. The similarities and differences are illustrated in Figures 1 and 2 which compare the mid- and near-infrared spectra of skin and hair. In the mid-infrared, strong amide I and II bands from keratin protein occur around 1660 and 1550 cm-1 for both skin and hair. Differences are seen in the intensities of the C-H stretching region (due primarily to lipid alkyl chains) and in the region below 1400 cm-1. In the near-infrared, the skin spectrum is dominated by the strong water bands at 1920 and 1440 nm, while the hair spectrum has strong protein bands at 1500, 2050 and 2180 nm in addition to the water bands, indicative of differences in the relative amounts of water and protein in the two tissues. Figure 1. Mid-infrared spectra of hair (top) and skin (bottom). The hair spectrum was acquired by diffuse reflectance on short pieces of hair mixed with KBr. The skin spectrum was acquired on a forearm in vivo by attenuated total reflectance using a 40?ZnSe internal reflectance element. (Spectra collected on a Bio-Rad FTS 40 FTIR.) Figure 2. Near-infrared spectra of skin (top) and hair (bottom). The spectra were acquired via a fibre optic cable on a grating spectrometer (LT Industries Quantum 1200). Although the functions and compositions of the SC and hair are similar, the structures of the two are very different. The SC is a stratified structure about 10-40 m m in thickness, with significant variability in composition between the deeper and surface layers. At the base of the SC, living cells extrude lipids and cornify into flattened, anucleate cells which are pushed outward until they are shed upon reaching the skin surface. Below the SC are the living epidermis, about 100-200 m m thick, and the dermis, about 2-4 mm thick (Figure 3). Figure 3. Diagram of a skin cross-section showing the multi-layered stratum corneum (SC), living epidermis and dermis. Adapted from D. M. Pillsbury, W. B. Shelley and A. M. Kligman, "Dermatology", W, B. Saunders Co., Philadelphia, 1956. Hair, on the other hand, is composed of a cylindrical cortex about 40-100 m m in diameter surrounded by an outer protective sheath of overlapping cuticle cells arranged like shingles on a roof. New cuticle at the base of the hair shaft consists of about 10 cell layers, altogether about 5m m thick. The cuticle cells gradually erode away over time as hair is increasingly exposed to UV radiation, detergents, and combing so that tip end hair may have few protective cell layers in the cuticle. Figure 4 shows SEM photomicrographs of new growth and weathered hair. The latter shows cuticle uplift, chipping and cracking, which result in a dull appearance of the hair and contribute to a rougher feel. Figure 4.  SEM photomicrographs of new growth (top) and tip end (bottom) hair. Longer exposure to sunlight, wind and grooming causes cuticle uplift, chipping and cracking. The tightly packed cortical cells, in turn, are composed of crystalline keratin, mostly in an a -helical arrangement, plus less organized protein structures. Figure 5 illustrates the substructure of hair. Figure 5 Lipids are an integral part of the membrane structures that act as a glue to hold the cuticle cells onto the cortex in hair. These lipids are primarily free fatty acids and cholesterol, but cannot be distinguished analytically from the sebaceous lipids which are always present. Sebum, in particular, affects foaming properties of shampoos, as well as oiliness and frictional behavior of the hair. In skin, lipid composition and structure are important in maintaining skin suppleness and elasticity and are also thought to be responsible for both the barrier to evaporative water loss and the water holding capacity of the SC. Water content in both skin and hair depends strongly on the relative humidity. Hair, in fact, acts as a hygrometer. Below 70% relative humidity (RH) water content in hair and SC is quite similar, about 10-30% by weight [1]. Measurements of skin tissue by electron probe analysis have indicated that water content within the SC gradually increases from about 10% to about 30% between the surface and deeper layers, followed by an abrupt increase in water content below the SC to about 70% water [2]. Changes in the water or lipid content of skin or hair may affect the feel or look of hair and skin by altering perceptions of dryness, elasticity or brittleness. Many of the products currently on the market or in development are designed to combat these problems and thus many of the spectroscopic measurements are geared toward evaluating these components. Spectroscopic Approaches to Skin and Hair Measurement 2. Attenuated Total Reflectance on Skin The desire to measure skin in vivo generally limits the number of approaches available for collecting spectroscopic data. ATR (attenuated total reflectance) has been applied extensively for infrared spectroscopy skin measurements because of its ease and rapidity of use. In ATR, a sample is placed on an infrared transparent crystal of high refractive index (the internal reflectance element or IRE), the geometry of which permits almost total internal reflection. The internal reflectance results in an evanescent wave which is attenuated in regions where the sample absorbs the radiation. The depth of penetration of radiation is quite shallow. For skin, it is calculated to be on the order of 1 micron in the mid-infrared, ideal for measurements of cosmetic treatment effects, where only the skin surface is of interest. ATR was initially proposed by Scheuplein as an alternative to reflection measurements on skin where scattering can be a problem [3]. Ferren later used ATR with a dispersive infrared instrument to measure products deposited on skin or hair [4]. He used untreated skin as the reference, so that he could look at product in situ, without interference from skin absorption bands. Photos show the author with his head directly on an ATR plate in order to measure a hair lotion and with his cheek pressed against an ATR plate to measure an aftershave lotion. The first ATR study to examine skin itself was performed by Puttnam and Baxter, using a V-shaped KRS-5 (a mixed crystal of thallium bromide iodide) ATR crystal to obtain two reflections off of the fleshy part of the hand [5]. They found only minor variations between the ATR spectra and transmission spectra obtained from thin slices of skin. The greatest variability between individuals was seen for the water band intensity around 3400 cm-1, attributed to perspiration effects. Concern about the toxicity of KRS-5 caused a switch to Irtran II (ZnS) [6]. The ATR technique was later improved by the use of a horizontal ATR crystal, making it possible for subjects to simply rest their arm on the IRE during data collection [7] (Figure 6). Figure 6. Photo of subject with her arm resting on a horizontal ATR accessory designed specifically for skin studies (Spectra-Tech). A drawback to ATR is that the depth of penetration of radiation into the skin is variable. The depth depends on the wavelength, the angle of incidence, and the refractive indices of the IRE and the skin. Treatment of the skin with lotions or hydration of the skin through increased relative humidity generally decreases skin’s refractive index, thus decreasing the depth of penetration of radiation. Hydration can also be induced by occlusion, such as that between skin and the IRE during the acquisition of an ATR spectrum. Because of the varied nature of the SC at different depths, spectra acquired before and after a treatment that affects the skin refractive index are not always comparable. In addition, the degree of contact between the skin and the IRE affects the spectrum. Baier found that application of creams or oils resulted in stronger amide I and II absorbances as contact between skin and IRE improved [7]. Klimisch and Chandra handled this problem by holding a damp towel to the skin before each measurement, ensuring reproducible contact [8]. In this case, they were looking at the substantivity of polydimethylsiloxanes on the skin rather than at hydration, so their approach was suitable. A common practice in skin studies is tape-stripping, where successive layers of skin are removed by scotch tape, followed by ATR measurements of the freshly exposed skin surface. Removal of layers of SC results in an increasingly hydrated surface, reducing skin’s refractive index and generally increasing the degree of contact between skin and IRE. Despite the uncertainties due to variability in refractive index and in degree of contact, ATR remains a valuable method of skin measurement as it is rapid and extremely simple to use. 3. Raman Spectroscopy on Skin Raman spectroscopy, with its relative insensitivity to hydration state of the SC, is increasingly popular for the study of skin. Raman spectra give information about protein conformation, lipid structures, disulfide bonds and amino acid side chain groups. The complementary nature of IR and Raman is illustrated by Barry, et al. who give assignments for transmission infrared and Raman scattering spectra of excised skin [9]. Assignments were also given in a later study by the same group comparing FT-Raman spectra of excised human stratum corneum, callus removed from the bottom of feet, plus hair and nails [10]. Although the spectra of the four tissue types were overall quite similar, some striking differences were noted. Stratum corneum tissue had a strong band around 2883 cm-1, attributed to methylene groups in lipid chains, which was mostly absent in the spectra of the other tissues. In addition, the hard keratins in hair and nails clearly showed the presence of more disulfide groups (510-530 cm-1) than appeared in the soft keratins of SC and callus. Raman data obtained in vivo and in vitro were compared using normal Raman scattering, a Raman microprobe and FT-Raman using near-infrared excitation [11]. The latter configuration was used to examine forefingers before and after tape-stripping. The most striking difference between the in vivo and in vitro spectra was the presence in the former only of a strong band near 3230 cm-1, believed to be due to N-H stretching. In addition, the in vivo spectra showed more intense C-C lipid skeletal backbone stretching vibrations between 1030 and 1130 cm-1. A comparison of the three Raman techniques indicate that significant differences occur in relative band intensities, probably due to orientation effects, but no differences in band frequencies. Raman spectroscopy using a confocal microscope represents a recent approach that holds a lot of promise for in vivo skin studies since the confocal microscope allows depth-profiling to be performed. In a recent paper, the use of a confocal Raman on different anatomical sites was described [12]. Spectra acquired at the skin surface indicated significant regional variation in SC composition such as between fingertips and forearms. 4. Near-Infrared Near-infrared diffuse reflectance spectroscopy has also been applied to skin, although this approach suffers from a poorly understood depth of penetration of the reflectance signal. Scheuplein discussed the optical pathways through skin from the UV to the infrared, and how the stratification of the SC enhances the possibilities of internal reflections [3]. Figure 7 shows possible pathways for light to interact with skin. Figure 7.  Optical pathways for light in skin: a) absorbance or transmission, b) regular reflectance, c) diffuse reflectance, and d) multiple internal scattering. Scheuplein assigned a value of 1.55 to the refractive index of the SC, and calculated a minimum of 4% regular (surface) reflectance based on the Fresnel equations. The off-normal angles of incidence produced by the irregular SC surface are expected to make the amount of regular reflectance higher [13]. Scattering from the skin surface is not appreciable below about 2000 cm-1, but is still significant in the near-infrared region and has been applied to studies of skin moisturization [14]. The lack of specificity of information regarding lipids and protein and uncertainty in the depth of penetration of radiation make this approach generally limited to information about water content and surface texture [15]. 5. Hair Hair, in contrast, is generally much easier to sample than skin as it is essentially in the same state whether it is attached to the scalp or not. Unless one is looking at the hair follicle, hair detached from a head differs only in that continual exposure to sebum is absent [See Section 1]. Thus, many spectroscopic approaches are available for examining hair. These include transmission, ATR, photoacoustic spectroscopy (PAS), infrared microscopy in the mid-infrared, Raman scattering and Raman microscopy, and near-infrared diffuse reflectance. A comparison of infrared microscopy, diffuse reflectance and ATR approaches for examining oxidative hair damage was made by Joy and Lewis [16]. ATR/KRS-5 was found to be the least reproducible method while diffuse reflectance spectra of fibres mixed with KBr produced very noisy spectra. Transmission spectra of flattened fibres allowed one to sample specific locations in a fibre and yielded better spectra. In another study, infrared spectra obtained using ATR with either a ZnSe or diamond IRE were compared to spectra from transmission microscopy in an examination of oxidative effects in hair fibres [17]. These authors also found the microscopy approach to yield good reproducible spectra. ATR/ZnS was found to be too irreproducible to be of value, although ATR/diamond, using 7-9 fibres at a time, was more succcessful. Although infrared microscopy is a popular approach for sampling hair fibres, it generally does not allow for high spatial resolution. The use of synchrotron radiation to achieve a spatial resolution of a few micrometers with infrared microscopy was described Bantignies, et al. [18]. The use of a high intensity source resulted in the ability to map across a fiber cross-section to study ingredient penetration into a fibre or oxidation effects at different depths. Confocal Raman microscopy offers another means to achieve high resolution, this time in three dimensions [19]. FT-Raman has also been applied to studies of hair and assignments for the Raman bands of hair are given in references [20] and [21]. Raman spectroscopy, even with a near-infrared laser source, is generally limited to unpigmented hair due to enormous fluorescence from melanin. Near-infrared reflectance spectroscopy allows bundles of hair rather than single fibres to be sampled and can be used in situ with a fibre optic probe. Ozaki, et al. used near-infrared reflectance to study water content in hair [22]. When data from large numbers of individuals are desired, spectroscopic measurements may be performed away from the laboratory. The advent of more portable instruments makes it possible to collect spectra in unexpected settings. Figure 8 shows the collection of near-infrared data in a salon. Figure 8.  Photo of near-infrared spectra being acqiured in a beauty salon. The spectrometer is a grating instrument from LT Industries, with a fibre optic cable.   Applications The main objectives of spectroscopic studies are generally different for skin and hair, although some overlap occurs. Skin studies are concentrated on product deposition and penetration, measurements of skin "dryness", plus an understanding of protein and lipid conformation. Most of the published studies on hair, on the other hand, concern oxidative effects of chemicals or sunlight. Below, the major areas of application for the spectroscopy of skin and hair in the cosmetic industry are discussed. 6. Measurement of Ingredients Applied to Skin and Hair As mentioned earlier, Ferren was among the first to use infrared spectroscopy to make measurements of living skin [4]. He demonstrated the ability to measure a variety of products deposited on the skin, including deodorant, hand lotion and an after-shave, by ATR. Also included were measurements of a hair preparation after application to the hair, requiring the subject to place his head onto the ATR crystal. Many of the other early infrared in vivo studies also focused on measurement of products deposited on skin. The substantivity of a skin cream on skin was demonstrated by Puttnam and Baxter, who noted increased infrared intensities of methylene stretching and deformation modes from the presence of fatty alcohols and mineral oil plus a weak feature at 1720 cm-1 due to stearic acid [5]. After washing, traces of the skin cream remained. Puttnam later studied the uptake of sodium dodecylbenzene sulfonate onto the palm of the hand [6]. Uptake was found to increase with either increasing surfactant concentration or with increasing time of exposure, but was decreased in the presence of an ether sulfate, presumably due to competition for sites on the skin. Somewhat more recently, ATR was used to look at the deposition and wash-off of siloxane polymers of differing molecular weights from skin [8]. Siloxane polymers are commonly used in skin care products because they impart a smooth, non-oily feel, and are non-toxic and non-irritating. The siloxane polymers are ideal for infrared analysis because they have a distinctive absorption band at 1261 cm-1. The amide II band of skin protein (at about 1550 cm-1) was used as an internal reference for degree of contact with the ATR crystal as it is not influenced by water content the way the amide I band (at about 1660 cm-1) is due to overlap with the water deformation band. A calibration curve was constructed by depositing known amounts of siloxane polymer onto the skin via a volatile solvent. After three soap and water washings, about 38% of the high molecular polymer was found to remain on the skin, while only about 9% of the lowest molecular weight polymer remained. The same group developed a different infrared method to determine the amount of siloxane polymers deposited on hair from a conditioner [23]. Diffuse reflectance was proposed as a simple and quantitative method, with the degree of dilution in KBr and grinding time optimized. A calibration curve was made using atomic absorption spectroscopic measurements, and a linear relationship between the two methods was found for siloxane polymer conce