Liu Lei,Yang Shuyan,He Wendan,Tang Ying

Department of Cosmetics,School of Science,Beijing Technology and Business University,China

Abstract

In this paper,instrumental analysis techniques for hair fibers and biological models for scalp were introduced as in vitro techniques applied for the efficacy evaluation of hair cosmetics.This review aimed to provide reference and guidance for the selection of appropriate in vitro methods to substantiate the performance of various hair cosmetics.The current status and development trends of in vitro evaluation technology for hair cosmetics were summarized and prospected.

Key words

hair cosmetics;in vitro methods;efficacy evaluation;hair fiber;scalp

Hair cosmetics include special used cosmetics for hair growth,hair dyeing,perming,and nonspecial used cosmetics for hair care.They are kind of frequently used commodity in people's daily life with different categories in terms of functions and mechanisms.In recent years,the hair cosmetic industry has developed rapidly in China and the local enterprises are facing fierce market competition.Consumers are becoming more rational and paying more attention to the efficacy of products.An efficacy claim without sufficient scientific support will negatively affect the marketing promotion of products.Therefore,it is an important issue that scientifically characterizing and evaluating the various performance of hair cosmetics nowadays.

Hair cosmetics can be divided into two groups according to the targeting area: hair fibers or scalp.For most hair cosmetics (functions including cleaning,repairing,bleaching and dyeing,shaping,etc.) that target on hair fibers,the efficacy evaluation mainly focuses on the changes in the appearance,structure,chemical composition and mechanical properties of hair fibers.By contrast,hair cosmetics (functions including anti-dandruff and hair growth promoting products) that target on the scalp,the efficacy evaluation is usually performed by applyingin vitrobiological models.

Hair cosmetics that target on hair fibers

Microscopic imaging

Scanning electron microscopy

Scanning electron microscopy (SEM) is a microscopic imaging technique capable of producing highly amplified and clear images of sample surfaces by scanning a object with a focused electron beam in vacuum.[1]SEM can directly observe the microstructural and morphological variations of hair fibers,which is the most commonly used method for micro-imaging analysis of hair.For example,the shape of scales on the surface of hair fibers is susceptible to light,weathering and combing,and there are obvious aging differences between hair root and hair tip.In 2010,Thibaut et al.observed the root,middle and tip part of a woman's 4.3 meter long hair with SEM,respectively.The scales of the hair root were well-formed,while the middle part showed lifted scales with irregular and rough appearance of the overall cuticle,and most of the hair scales in the tip part had fallen off,which directly exposed the hair cortex.[2]In addition,SEM can be used to assess the impact of hair cosmetic treatment on the hair microstructure,which facilitates the evaluation of the damaging or repairing function qualitatively or semi-quantitatively.Ahn et al.of Yonsei University in Korea[3]reported a study about SEM on hair damage associated with permanent hair dyes in which the scales of dyed hair became fragmented,peeled and detached.In 2012,Fernández et al.[4]have found that hair conditioner agent prepared by artichoke extract can improve the lifting of hair scales in the aging process thus supporting its hair protection effect.

Transmission electron microscopy

Transmission electron microscope (TEM) is a microscopic imaging technique which projects electron beam onto very thin samples and uses electrons to collide with atoms in substances to produce solid angle scattering and imaging.TEM can be used to observe the ultrastructure of inner cross section of hair fibers.In the above research of Ahn et al.,TEM is also used to observe the damage of hair cuticle caused by permanent hair dye,which appears to be most severe in the first 6-24 hrs and it will take 8 weeks to restore to the undyed state.Therefore,it is suggested that an interval for coloring hair of at least 8 weeks is desirable to prevent accumulated damage.[3]Similarly,Richena et al.have reported that using TEM to assess the damage to hair's microstructure during ultraviolet radiation and shampooing and have found that excessive illumination and cleaning can make hair being damaged easily.[5]Kim et al have found that the addition of procyanidin oligomers to hair care products can slow down the degradation of melanosomes and proteins in hair cortex during bleaching and dyeing.[6]

Atomic force microscopy

Atomic force microscopy (AFM) obtains the nano-scale morphological information of a substance via interactions between the probe and the measured sample.AFM has unique advantages in local 3D topological imaging owing to its ultra-high image resolution,ease of testing and sample preparation (not require gold coating and high vacuum conditions as for SEM and TEM).In the past 20 years,AFM has been widely used to observe various micro- and nanostructures (such as layer A,epidermis,endothelial cells and cell membrane complexes) in the hair cuticle,and the data are used to compare the effects of environmental factors and cosmetic treatment on the roughness and tribological properties of hair,so as to substantiate the hair care efficacy.For example,Chen et al.[7]have employed different modes of AFM to study the relationship between the distributionthickness of conditioner and hair friction properties.AFM can also be used to characterize various physical properties of hair fibers,such as friction,adhesion,wear,elastic stiffness and viscoelastic nano-mechanical properties.In 2013,Smith et al.have used the AFM images to determine the microscale friction coefficient,tip-to-surface adhesion force and the corresponding adhesion energy of hair surface substructure.[8]In another study,the Young's modulus of hair fiber has been determined by AFM,and the modulus of hair surface,with a decreased elasticity,has been found to decrease after bleaching treatment.[9]

Optical coherence tomography

Optical coherence tomography (OCT) can also be used for high resolution image analysis of the cross section of hair fibers.OCT is a novel imaging technique based on the principle of low length coherent interferometry to obtain biological structure images.[10]In 2014,Camacho-Bragado et al.have reported that using X-ray tomography to investigate hair breakage of African-American women and have found that when the strain of hair fibers exceed 30%,the cross-section area (especially where the breakage occurred) will reduce dramatically.[11]In addition,OCT can analyze the structural and morphological changes of hair caused by external factors in an identical area,which has the advantages of comparing the changes in repeated testings accurately with reduced measurement error.In 2016,Velasco et al.have reported the use of OCT to investigate the ultrastructural change of hair caused by oxidized hair dyes.This study has showed that the scattering coefficient and refractive index of hair will increase after dyeing,while the clarity of OCT images of hair medulla and cortex will decrease.as thus,judging the damage of medulla and cortex structure.[12]

Spectral analysis

Spectral analysis is used to determine chemical composition and modification of substances according to their optical properties.UV-Visible spectroscopy,fluorescence spectroscopy,infrared spectroscopy and Raman spectroscopy are usually employed.

UV-Visible spectroscopy

UV-Visible spectroscopy includes diffuse reflectance spectroscopy and absorption spectroscopy in the ultraviolet-visible region (320-780 nm).The diffuse reflectance spectrum refers to the reflectance of a substance in the ultraviolet visible range,and is often used to determine the color and brightness.By converting the reflectance values to the CIELab color space,diffuse reflectance spectroscopy can be used to quantitatively evaluate the gloss and color change of hair fibers following hair dyeing and hair care treatment.In 2016,Tang et al.[13]have reported the color changes of different types of hair samples from Chinese,including white/black hair,gray hair and bleached hair during UVA photoaging by diffuse reflectance spectroscopy.Different hair types shows significant different sensitivity to UV radiation and photochromic response.Natural white and gray hair fibers exhibit the usual photoyellowing while blackdyed and bleached hairs show photofading and additional photobleaching,respectively.Dario et al.[14]have found that the combination of pomegranate extract and hair conditioner can effectively restore the color of dyed hair during photoaging.On the other hand,absorption spectrum reflects the absorption characteristics of chromophores in the visible wavelength range,and can be used for quantitative analysis of dyes or absorbents.In 2014,Pires-Oliveira et al.[15]have analyzed the absorption spectrum of hair extract solution and have compared the hair damage caused by four surfactants.It has been found that the absorption peak area is proportional to the melanin particles and protein components degraded from hair.A spectrophotometric method has been established to characterize the hair damage.

Fluorescence spectroscopy

Fluorescence spectroscopy is used for qualitative or quantitative determination of substances by identify fluorophores.Amino acids in hair keratin such as tryptophan,tyrosine and cysteine are fluorescing species.Therefore,the degradation of protein components in hair and the protective effect of functional components in hair care products can be investigated by fluorescence spectroscopy.In 2012,Estibalitz et al.[16]have found that the photodamage of hair can be quantified by monitoring the fluorescence intensity of tryptophan in hair and hair conditioner prepared from rice extract has photoprotective effect on hair.[4]Furthermore,in contrast to the quantitative detection of tryptophan by twodimensional emission spectroscopy,three-dimensional fluorescence spectroscopy can be used to obtain fingerprints so as to analyze various amino acids and their oxidation products in hair fibers to after different cosmetic treatments.[13]

Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) is generated by the transition of the molecular vibrational energy level of the compound after absorption of infrared light,and contains structural information such as functional groups and crystalline states of the compound.By connecting FT-IR with ATR attenuated total reflection,hair fibers can be directly measured,and the fingerprint characteristics of infrared spectroscopy can be used to evaluate the effects of different cosmetic treatments (such as moisturizing,bleaching and dyeing) on the structure and morphology of hair keratin.[17]In 2016,Tang et al.[13]have analyzed the hair samples after different bleaching and dyeing treatments by FT-IR 3D fingerprinting and have found that UVA and hydrogen peroxide can break the disulfide bond of cystine in hair and produce cysteine which can be easily oxidized to produce a large amount of sulfoalanine.Since the cystine content on the hair surface is relatively constant (about 11% to 18%),sulfoalanine can be used to monitor the degree of oxidation of high-sulfur proteins.

Raman spectroscopy

Raman spectroscopy (RS) is a technique for qualitative and quantitative determination of the composition and molecular structure of a substance by using inelastic scattering spectrum (“Raman effect”) produced by vibration of a compound after irradiation with light.Raman spectroscopy of hair fibers can reflect the skeleton vibration of the main chain of protein,the conformation and existence of side chains of amino acids,and the minor changes caused by changes in external environment (S-S conformation and secondary structure of protein molecules,etc.).[18]From 2006 to 2010,Kuzuhara A et al.have investigated the influence of aging and bleaching on hair melanin content,protein fiber structure and tensile strength.[19,20]By analyzing the Raman spectra of cross-sections at different depths of different ages and treatments (bleached hair or black hair),they have found that melanin granules in the hair cortex area will decrease with age.[21]The content of disulfide bond decreases from the stratum corneum to the cortex,while the cysteine content increases.In addition,oxidative agents from bleaching powder also promote the decomposition of melanin particles in hair cortex and result in a higher level of S-S decomposition in black hair during bleaching.[22]

Chromatography

Chromatography is a technique for separating and analyzing substances according to the difference of distribution coefficients between stationary phase and mobile phase.Tryptophan is often used to quantitatively evaluate the loss of protein components in evaluating the damage degree of hair caused by cosmetics,but tryptophan quantification by fluorescence spectrophotometry is vulnerable to other fluorescent substances in the detection system.In 2017,Dario et al.[23]have developed a high performance liquid chromatography (HPLC) with fluorescence and UV detection.This is a method for quantitative determination of tryptophan and photooxide canine uric acid after alkali hydrolysis of hair samples.The results show that the bleaching and dyeing process will lead to the loss of protein/amino acid,but tryptophan will not degrade in a large amount during hair photoaging.Khumalo et al[24]have analyzed the chromatographic analysis of the hair hydrolyzed by hydrochloric acid and have combined with amino acid analyzer.It has been found that the contents of arginine,citrulline and cystine in damaged hair decrease,while the levels of glutamine increase.Besides,chromatography is often combined with mass spectrometry for quantitative detection of functional or prohibited components in cosmetics.

Mass spectrometry

Mass spectrometry (MS) is an analytical technique that can quantify different molecules according to their mass-charge ratio.Keratin and keratin association protein (KAP) provide important structural and mechanical properties to hair fibers.Proteomic mass spectrometry can be used to characterize the effects of different cosmetic treatments or environmental factors on the protein content of hair fibers by identifying the molecular modifications of hair keratins and amino acid groups in major KAP.In 2013,Dyer et al.[25]have used LCMS/MS to identify changes of protein content in hair samples,before and after alkali treatment or bleaching.These results show that alkali treatment slightly increases the degree of oxidative damage in hair,while the bleaching treatment leads to significant oxidative damage to the hair keratin (mainly cysteine).In 2016,Tang and Dyer have analyzed the hair samples from frequent hair dyes in China.It has been found that the oxidation level of hair dyed protein is nearly 1.6 times higher than that of undyed hair,so the often dyed hair is more susceptible to sunlight damage.[26]

Thermal analysis

Thermal analysis is a technique to characterize the physical properties of substances with changing temperature under programmed temperature control.It includes thermogravimetric analysis (TG),derivative thermogravimetric analysis (DTG),differential thermal analysis (DTA) and differential scanning calorimetry (DSC).DSC is often used to determine the denaturation temperature and enthalpy of keratin.[27,28]In recent years,DSC has been increasingly used to evaluate the efficacy of hair care products.In 2011,Zhou et al.[29]have evaluated the degradation of hair keratin and the repair effect of cosmetics on damaged hair by DSC.The results show that the denaturation temperature and denaturation enthalpy change of keratin decrease significantly with an increase of the treatment temperature from 205 ℃ to 232 ℃,and the degradation of keratin become more serious.The thermal degradation of keratin and cortex damage can be effectively reduced by using polymers (lauryl methacrylate copolymer,polyquaternary ammonium salt-55,etc.) to treat hair.In 2016,Popescu et al.have found that when the denaturation and degradation of alpha-keratin occur in dry hair,the DCS denaturation temperature of wet hair decreases.It has been suggested that DSC should be combined with XRD and stretching test to better understand the actual effect of cosmetics on hair.[30]

Mechanical performance analysis

The mechanical properties (tensile and torsional properties) of hair fibers are determined by the alphakeratin and disulfide bonds in the hair cortex.At the same time,the mechanical properties of hair are also affected by various environmental factors: an increase in the relative humidity or temperature usually results in the decrease of Young's modulus and the increase of ductility,while the distortion will cause damage to hair fibers,resulting in the decrease of fracture stress,fracture strain and Young's modulus.[31]Mechanical properties such as stretching of hair can be measured by Instron INSTRON series universal material tester,Dia-Stron MTT tensile tester,etc.Test parameters include stretching strength,fracture stress,strain at break curve,elongation at break,etc.[29-32]Davis et al.[32]have reported that a formula containing caffeine,nicotinamide and panthenol has the effect of enhancing hair tension and flexibility.The tensile and torsional properties of hair fibers under external forces are also related to their flexibility.Besides hair care products,they can also be used to evaluate hair styling products through tensile and torsional tests.For example,Liu et al.[33]have evaluated the shape memory effect and wash resistance of a polyurethane polymer coated hair by measuring its mechanical properties.

Combing performance

Friction coefficient and elastic properties of hair fibers are important physical parameters in hair combing performance.Hair scales composed of lipids (18-methyleicosanoic acid) and keratins on the surface of hair stem are closely related to the tribological properties of hair.When the hair is aged or damaged,the lipid is lost,the scales become lifted and peeled off,and the surface of the hair becomes rough,the friction coefficient increases,and hair becomes difficult to comb or knot easily.Therefore,characterizing the tribological properties of hair surface is very important for quantifying carding performance and evaluating the efficacy of shampoo and hair care products.Chevalier[34]has obtained the self-friction coefficient equation for calculating a single hair through a nylon thread test,and has measured the static friction coefficient of wet hair significantly higher than that of dry hair.It has been also found that alkali treatment will cause irreversible increase of hair friction coefficient.There are hairspecific testing instruments to facilitate the evaluation of carding performance of cosmetics,such as Dia-Stron MTT series testers and Ashland Aqualon SLT components.Da et al.[35]have used Dia-StronTM (MTT175 Miniature Tensile Tester) to analyze the after-dyeing protective effects of different hair conditioners on hair and the formulations containing alkanetriol and panthenol show usefulness to reduce the combing resistance of hair.

Hair cosmetics that target on the scalp

For hair growth and anti-dandruff cosmetics that target on the scalp,the efficacy evaluation is performedin vitrousing cultured cells,bacteria and hair follicle tissues.

Cultured cells

Dermal papilla cells,keratinocytes and hair follicle epithelial cells are often applied to evaluate the efficacy of hair growth promoting products.The effect of functional components on hair growth promoting is measured by the promotion of cell proliferation,changes of cytokines (i.e.VEGF) and related gene expression.Rho et al.[36]have reported that Costunolide (a sesquiterpene extract from the root of Asiasari radix) can stimulate the proliferation of immortalized human scalp hair follicle dermal papilla cells (hHFDPC),which has been confirmed in further animal experiments,indicating that this component has significant hair growth promoting effects.Immortalized human keratinocytes (HaCaT) and dermal papilla cells (DP) have been widely used in the screening of hair growth promoting and antialopecia components.A variety of plant extracts and synthetic chemicals have obtained the same resultsin vitrocells and further animal experiments.[37-39]In addition,the production of white hair is believed to be related to the destruction of melanin by high oxidative stress,Sextius P et al.have reported that the primary human melanocyte model damaged by hydrogen peroxide is used to evaluate the efficacy of black hair.Polygonum multiflorum extract has been found to effectively inhibit the increase of intracellular ROS level and apoptosis.,inhibiting the decrease of melanin under oxidative stress,and thus playing an important role in black hair.[40]

Bacteria

Malasseziahave been closely associated with the dandruff.The fungus on the scalp is mainlyMalassezia,among whichM.restrictaandM.globasoare dominant species.[41]Therefore,theMalasseziabacteriostatic test is widely used to evaluate the efficacy of anti-dandruff shampoo with antimicrobial ingredients.Turner et al.[42]have reported the anti-dandruff effect of shampoo which containing different antimicrobial ingredients (such as piroxone ethanolamine,ketamine and zinc pyrithione) according to the size ofMalasseziainhibition zone and human experiments.In addition to comparing different formulation products,Malasseziatest is also widely used in screening of antimicrobial and antidandruff materials.Several literatures[43,44]report that plant extracts (such as tea tree oil and asparagus root extracts) with good antimicrobial and antidandruff properties are screened by the determination of minimum inhibitory concentration (MIC) ofMalassezia.These plant components can be used as natural substitutes for chemical antimicrobials in antidandruff cosmetics.

Tissue models

Tissue models include human or animal hair follicle culture systemin vitroand commercial 3D skin models.Compared with cell lines,tissue model is closer to the real human body in threedimensional structure and physiological function,and is generally considered as having higher reliability in efficacy evaluation.Therefore,in recent years,in vitrohair follicle culture systems have been widely used in research and development of hair growth promoting products.Park et al.[45]have usedin vitrocultured human hair follicles to study the effects of bis-oleamido isopropyl alcohol (BOI) on hair growth promoting.It has been found that BOI treatment can promote hair growth in cultured human hair folliclesin vivoand induce earlier conversion of telogen into anagen.In addition,the tissue model can be co-cultured with exogenous microorganisms to evaluate the efficacy of antimicrobial and anti-dandruff.In 2013,Turner et al.have reported thatMalasseziagrow in culture on a surface of VitroSkinTMskin model,and has found that shampoos with anti-microbial ingredients (zinc pyridinone and climbazole) had good antidandruff effect.[42]

Summary

Compared with traditional animal and human experience,in vitroevaluation methods such as biophysical instrumental analysis andin vitrobiological model are more economical,simple and rapid.It has important practical significance for screening functional ingredients,studying the mechanism and optimizing the formulation.Nondestructive or micro-destructive instrumental analysis has been used to evaluate the changes of hair fibers in color,brightness,microstructure,protein loss and mechanical properties after cosmetic treatment.Hair cosmetics that target on the scalp,the efficacy evaluations are mainly performed by employingin vitrobiological models such as skin cells,microbial models and hair follicle tissues.

Acknowledgements

The work was supported by the National Natural Science Foundation of China (51403006) and the Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13thFive-year Plan (CIT＆TCD 201704043).