Wang Huan, Pan Yao, Zhao Hua

Beijing Technology and Business University, China

Abstract The performance and formation mechanism of sensitive skin were summarized. Various evaluation methods for soothing skin cosmetics such as physical and chemical methods, biochemical methods, cell biological methods, three-dimensional recombinant skin model alternative method, animal experiment method, subjective evaluation, semi subjective evaluation and objective instrument evaluation method were introduced. The new idea of soothing efficacy evaluation was proposed and the development direction was put forward.

Key words soothing cosmetics; sensitive skin; efficacy evaluation

With the improvement of consumption level,people pay more and more attention to the skin health, and the attention to sensitive skin is increasing year by year. Factors such as climate change, air pollution and the complexity of cosmetics types and components increase the probability of sensitive skin and the possibility of skin irritation. Developers in the cosmetics industry aware of this phenomenon,putting products for sensitive skin in an increasingly important position, and there are more products on the market that claim soothing effects.

Formation mechanism of sensitive skin

Sensitive skin refers to a highly reactive state of the skin under physiological or pathological conditions,mainly in the face. The clinical manifestations are that the skin is prone to subjective symptoms such as burning, pruritus and tension when stimulated by physical, chemical, mental and other factors, with or without erythema, scales, telangiectasia and other objective signs.[1]With the aggravation of environmental pollution and the increase of mental pressure and other reasons, the incidence of sensitive skin is gradually increasing, and it attracts more and more attention.

At present, it is believed that the occurrence of sensitive skin is a complex process involving skin barrier, neurovascular and immune inflammation.Under the interaction of internal and external factors,the function of skin barrier is damaged, resulting in the increase of sensory nerve afferent signals, the enhancement of skin response to external stimuli, and skin immune inflammatory response. If a cosmetic claims to solve these skin problems, how is to build scientific support for it?

In vitro experiment

In vitro experimental methods include physicochemical method, biochemical method, cell biology method, three-dimensional recombinant skin model alternative method and so on.

Physicochemical methods

Physicochemical method is a convenient method to evaluate the effect of cosmetics by detecting its physical properties and specific chemical components. At present, majority are qualitative and quantitative detection of specific efficacy components in the evaluation of cosmetic soothing efficacy.

Ceramide, N-fatty acyl sphingosine, is an important component of human skin lipid. It has the functions of moisturizing, maintaining the stability of cuticle structure and damage repair. Its nature and quantity affect the stability of skin barrier and the occurrence of some skin diseases. Currently, there are more systematic analysis methods such as cuticle sampling, liquid chromatography, ionization spray mass spectrometry (ESI-MS), atmospheric pressure chemical ionization mass spectrometry (APCI-MS),tandem mass spectrometry (MS-MS) and so on.[2]

Biochemical methods

Biochemistry is a method to test and analyze the characteristic indexes (e.g. free radicals) and important biomolecules (e.g. proteins) which affect the efficacy by means of spectral analysis,electron microscope and other physical and chemical techniques. The biochemical experiments commonly used in the evaluation of anti-inflammatory and antisensitive efficacy include hyaluronidase inhibition test and free radical scavenging test, and some researchers use erythrocyte hemolysis test as well.

Hyaluronic acid (HA) can regulate the secretion of cytokines, and affect the adhesion, growth,proliferation and differentiation of cells. Therefore, it plays an important role in maintaining skin moisture and elasticity, wound healing and angiogenesis.[3]HAase is a specific lyase of hyaluronic acid, inhibiting the activity of HAase is to ensure the normal content and function of HA. Therefore, the anti-sensitive activity of the substance can be evaluated by using the inhibition rate of HAase as an index. Hu Guosheng established an anti-inflammatory response model in vitro by hyaluronidase activity inhibition test, and studied the inhibitory and anti-inflammatory effects of three plant extracts of cactus, chrysanthemum and asiatica on hyaluronidase activity.[4]

Free radicals are produced during mitochondrial electron transport, which attack cell membranes,proteins, and DNA,[5]inhibition of free radical activity can effectively alleviate the degradation of cell structure and function caused by external stimulation-induced excessive free radicals, and then maintain the normal barrier structure of the skin. Free radical scavenging experiments include 1,2-diphenyl-2-picrylphenylhydrazine radical (DPPH·), hydroxyl radical (·OH), superoxide anion radical(·O2-), nitric oxide radical (NO·) and other substrates. It is used to detect the antioxidant effect of various small molecular antioxidants, including enzymes, vitamins,polyphenols, and flavonoids.

Cell biology method

Cell biology is the science that studies the structure, function and life cycle of cells. Cell biology methods establish the cell models, simulate the human physiological environment in vitro to culture cells, and measure the changes of cells before and after contact with the test substances to verify the effectiveness of the raw materials.

At present, the skin barrier structure-related proteins have not been widely analyzed and studied,but they have gradually attracted the attention of the biomedical community. In recent years, there have been studies on the effect of external active substances on the expression of AQP-3 (aquaporin-3),FLG (filaggrin) and Caspase-14 (cysteine aspartate specific protease-14) in keratinocytes.[6-8]When the cells are in an inflammatory or high osmotic environment, the expression of AQP-3 in the cells is up-regulated, so the expression of AQP-3 is higher in the skin of aging or atopic dermatitis.[9]

Keratinocytes from basal layer, spinous layer, and granular layer to cuticle are the dynamic process of cell proliferation, differentiation, movement, death and shedding. Tape stripping test is used to understand the morphology and function of keratinized envelope(CE) — an important structure of skin barrier by collecting exfoliated keratinocytes.[9]The commonly used method is that the skin scales are collected by adhesive tape, and the maturity of CE is judged by fluorescence staining to evaluate the damage degree of skin barrier.[10]

The protective ability of skin nerve endings is weakened, the density of nerve fibers is increased and the reactivity of sensory nerves is increased.The interaction of the three can cause the dysfunction of skin sensory nerves, which is related to the occurrence of transient receptor potential (TRP) family activation.[11]In recent years, vanillic acid subtype transient receptor potential 1 channel protein(TRPV1), which is a research hotspot in cosmetics industry, can be activated by physiological or subphysiological temperature (lower than the normal activation temperature of TRPV1), expressed in fibroblasts, keratinocytes, melanocytes and mast cells, resulting in sensitive symptoms such as itching, burning, or pain.[12]Nowadays, most of the cosmetic raw materials developed for neurogenic skin sensitivity are TRPVl expression inhibitors or functional antagonists, through the detection of TRPVl expression, we can evaluate that whether the active substance has the anti-allergic effect of relieving facial discomfort.

Three-dimensional recombinant skin model alternative method

The alternative method of three-dimensional recombinant skin model is a method of using tissue engineering technology to culture human skin cells on a special inserted petri dish to construct an artificial skin tissue model with three-dimensional structure,which is used to replace the animal model to carry out experiments.[13]Three-dimensional recombinant skin model provides a new technical means for the evaluation of safety and efficacy of cosmetics.

The three-dimensional recombinant skin model has a skin structure that is highly similar to human skin. It can be used not only for the safety test of cosmetics, but also for the study of skin absorption and action mechanism of effective raw materials in formula products, as well as for the analysis of the interaction between different cells, providing the comprehensive information of raw materials or formulated products from safety to efficacy. The main advantages of using skin model to evaluate the efficacy of cosmetics include: a complete cuticle layer, different physical and chemical properties of the test substance (hydrophobic substances or solids,mixtures or formula) can be applied to the skin models; accurate and controllable test conditions,easy to quantify data and good repeatability; shorter test period, the time and economic cost are reduced compared with animal and human experiments; the use of experimental animals is effectively reduced.

When the skin model is used to evaluate the soothing effect of cosmetics, it can be applied to the epidermis or whole skin model stimulated by surfactants or microorganisms, etc. The antiin flammatory repair ability of cosmetic efficacy raw materials or formulations was evaluated by detecting the effect of cosmetics on the vitality of skin model tissue, the secretion of inflammatory chemokines(PGE2) and cytokines (such as IL-1α, IL-8 and TNF-α, etc.), and the expression of related genes.Currently, although the skin model highly simulates the structure of human skin and provides scientific support for the study of the mechanism and effect of the tested substance on the skin, there are still some shortcomings in the commercial skin model. Due to the lack of hair follicles, sebaceous glands and other skin appendages, a certain gap still existes between the model and the real skin.[13]

In vivo experiment

In vivo experiment mainly includes animal experiment method and human test method, in which human test method can be divided into subjective evaluation method, semi-subjective evaluation method and objective evaluation method.

Animal experiment method

Animal experiment method is the most widely used method to evaluate the anti-sensitivity effect.Animals are used as the subjects, after patch excitation, the researchers evaluate the anti-sensitivity effect of the samples according to the damage of animal skin. In addition to patch stimulation test,most of them are anti-inflammatory and allergic reaction experiments, but the research is not only limited to the skin, but also involves the impact on the immune system of the body. The related experimental observation indexes include histopathological observation, determination of various cytokine levels and analysis of related gene expression, as well as detection of eosinophil, leukocyte and mast cell activity.[14-16]

In the study of an effective skin care product containing portulaca oleracea extract, He Li et al. evaluated its anti-inflammatory and anti-sensitivity effects by constructing a mouse auricle swelling model induced by xylene.[17]Wang Ling et al. verified the antiallergic and antipruritic effects of radix ophiopogonis,sophora flavescens, tribulus terrestris and artemisia annua by histamine phosphate-induced pruritus in guinea pigs and low molecular dextran-induced pruritus in mice.[18]Zhang Jingxia's study showed that mice with AQP-3 gene knockout had disorders in skin moisturizing, elastic maintenance and barrier repair, and glycerol administration alleviated these symptoms.[19]

On November 7, 2002, the European Parliament and the Council of the European Union reached an agreement in Brussels, which decided to “prohibit the use of animals for toxicity and allergy tests within the European Union from 2009, to prohibit the use of animals for safety assessment of cosmetic raw materials in 2013, and to gradually prohibit its member countries from importing and selling cosmetics for animal experiments from abroad”. The application of animal experiment in the development of cosmetic raw materials has begun to be gradually replaced in the world.[20]

Human experiments

In the development of most anti-sensitivity cosmetics raw materials and products, human experiment is an indispensable part. It mainly includes subjective evaluation method, semi-subjective evaluation method and objective evaluation method.

Subjective evaluation method

Subjective evaluation includes visual assessment and subject self-assessment. Visual evaluation is usually carried out qualitatively or graded by experts or doctors on the skin state of the subjects, such as skin color, skin elasticity and so on, and the selfassessment of the subjects is usually carried out in the form of questionnaires. The indexes include pruritus, tingling,burning and tension, and the subjects score their skin sensitivity according to the questionnaire.[21]

The commonly used subjective evaluation methods of anti-sensitivity efficacy include visual analog scoring method, sensitivity index evaluation scale,and investigator's global assessment (IGA) score.[22]Li Yong et al. used a randomized double-blind self-controlled trial to observe the clinical effect and safety of 25 patients after pulsed laser surgery with soothing moisturizing cream containing purslane.The visual evaluation simulation method was used to observe and evaluate the pain, edema, erythema subsidence and scab removal time of each subject on the treatment side and the control side, and to observe the adverse reactions such as pigmentation. Each patient was observed by the same staff.[23]In order to investigate the efficacy and safety of soothing special facial cream in the treatment of facial sensitive skin,Li Yun et al. used a sensitivity index evaluation scale method, which was a new scale to test the degree of skin sensitivity. The scale used a 10 cm visual analogue scale to evaluate the overall sensitivity of the subjects to their skin. Then subjective (skin irritation, tingling, burning, heat, tension, itching,pain, overall discomfort, flushing) and objective(redness, desquamation, edema/ swelling, exudate,scabbing) of 14 sensitive skin symptoms were evaluated. Each symptom was scored on a scale of 0 to 10, and the individual-specific sensitivity index was added, followed by statistical analysis.[24]

The human stimulating patch test is an indispensable part in evaluating the efficacy of soothing cosmetics.Usually, volunteers aged 18 to 60 are selected as the subjects to smear stimulants and samples, and scored according to the subjective feelings of the patients,so as to evaluate the anti-sensitivity effect of the samples.

Because of its subjective factors, the subjective evaluation method cannot objectively characterize the soothing effect of cosmetics. It is necessary to combine with the objective instrument evaluation method to study the correlation between the subjective evaluation results and the objective instrument detection results, and then to characterize the soothing effect of cosmetics in a more comprehensive way.

Semi-subjective evaluation method

Semi-subjective evaluation method has been widely used in the determination of sensitive skin,such as lactic acid tingling test and capsaicin test.

Lactic acid tingling test (LAST) is one of the widely used evaluation methods. LAST designed by Frosch et al. has been considered as the most effective method to evaluate sensitive skin. The most classic of which is the smearing method: at room temperature,5% or 10% lactic acid solution (50 μL) is applied to nasolabial sulcus and either side of cheek, and the subjects are asked about their conscious symptoms at 0, 2.5, 5 and 8 min, respectively. Score is according to 4 points (0 for no tingling, 1 for mild tingling,2 for moderate tingling, and 3 for severe tingling).[25]Then the two scores are added together, and the total score more than 3 is positive for lactic acid tingling reaction.[26]When the skin barrier is damaged and lactic acid enters the skin, unmyelinated class C nerves are stimulated, resulting in a tingling sensation.[27]This method is simple, effective and reproducible, and is widely used in clinical detection and experimental research.

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Capsaicin test is a commonly used method to evaluate sensory nerve sensitive skin. Jourdain et al.used five different concentrations of capsaicin to apply locally to the nasolabial sulcus to explore the optimal concentration of capsaicin for facial nerve sensitivity, and further detected the sensitivity of different ethnic facial nerves with different concentrations of capsaicin. The role of capsaicin in the evaluation of nerve sensitivity skin was confirmed.[28]The specific method of operation is to place the two-layer filter paper with a diameter of 0.8 cm at about 1 cm outside the nasolabial groove on one side and on either side of the cheek. Place 50 μL of capsaicin with a content of 0.1% on the filter paper and ask the subjects how they feel (1 is barely perceptible, 2 is mild perceptible, 3 is moderate perceptible, 4 is severe perceptible and 5 is pain). If the duration of burning sensation is more than 30 s and the degree of burning sensation is more than 3 points, the subjects are positive.[29]

Sodium dodecyl sulfate (SLS), as a classical skin irritant, is an anionic surfactant that is easy to penetrate into the skin. The basic method of SLS skin irritation test is to place a certain concentration of SLS in the patch chamber and wrap it locally in the test site. 24 hours later, the chamber is removed.The skin changes of the test site are observed and the clinical score and non-invasive instrument detection are carried out at specific time points after removing the patch chamber.[30]

In addition to the common chemical probe stimulation test described above, dimethyl sulfoxide(DMSO), chloroform-methanol mixture and menthol can also be used as chemical probes, and the sensitivity of the skin is evaluated by the skin irritation response to the chemical probes.[30]

Objective evaluation method

Objective evaluation method is a method to collect,sample analysis and statistical analysis of the skin state parameters of the subjects before and after the use of cosmetics, and then to evaluate the efficacy of cosmetics.

1) Skin water dispersion loss: transepidermal water loss rate can be used to detect the amount of skin cuticle water dispersion loss. The commonly used test instrument is Tewameter instrument, and the unit of test parameters is g/(h ·m2). TEWL is related to skin barrier function, skin water content and test environment. Zhu Xuejun et al. found that TEWL was not significantly correlated with gender, but with age, with the highest in neonates and the lowest in the elderly. When the skin barrier function is destroyed,the TEWL value increases.[31]

2) Skin moisture content: the structure of skin cuticle is dense, and the water content is less than other tissues, forming a layer of natural water barrier to prevent water loss. The normal stratum corneum hydration (SCH) is maintained in a certain range.The proper wetness of cuticle is a prerequisite for maintaining the physiological environment of the skin and promoting skin metabolism. The commonly used test methods are skin capacitance or skin conductance method, its test instruments include Corneometer capacitance tester and Skincon conductance tester.When the skin barrier function is damaged, SCH is insufficient and water is lost through the skin, which makes the skin drier.[32]

3) Skin erythema index: the skin erythema index can directly reflect the hemoglobin in the dermal papillary layer. The increase of erythema index indicates that the vascular reactivity of the skin is high,which is one of the manifestations of sensitive skin.Therefore, by testing the erythema index of the skin in contact with the subject, it can indirectly reflect the degree of allergy of the skin. The skin erythema index can be measured by DermaSpectrometer,Erythema Meter and other reflectance spectra,VISIA standardized multi-light source image acquisition instrument, or Chroma Meter instrument in trichromatic analysis. Chroma Meter instrument adopts the chromaticity system specified by Commission Internationale de L'Eclairage (CIE) to detect the reflected light on the surface structure of the skin. The L*in the L*a*b*chromaticity system is used to represent the blackness and whiteness, and a*is used to represent the redness and green. b*stands for blue and yellow chromaticity. The comprehensive evaluation of the three parameters can reflect the change of skin chromaticity, which can be expressed in the form of quantitative value. This can accurately re flect the change of skin erythema, so as to evaluate the irritation degree of the test material to the skin.[33]

4) Skin surface acidity and alkalinity (pH): the skin surface pH is formed by water-soluble substances in the cuticle, sweat discharged from the skin, waterfat emulsions on the skin surface, and carbon dioxide discharged from the skin. Generally maintained between 4.5 and 5.5, weakly acidic. The increase of pH value on the skin surface can directly or indirectly affect the metabolism of keratinocytes, resulting in abnormal proliferation and differentiation of keratinocytes,changes of skin barrier function and decrease of cuticle compactness. The higher the pH value of the skin, the lower the water permeability barrier function.[34]

5) Skin sebum secretion: skin sebum secretion is measured in addition to the skin sebum meter, and is often tested using the photometer principle. Excessive sebum will affect the normal arrangement of cuticle lipids, but also affect the integrity of the skin barrier.In addition, excessive sebum provides conditions for the reproduction of microorganisms such as propionibacterium acne, which in turn impairs skin barrier function.[35]

In addition to the above test indicators, other physiological index of the skin test, skin elasticity,skin thickness and skin surface structure changes can be used to judge the sensitivity of the skin as well. This objective evaluation method applied to the human body, can provide more intuitive data results,and the process is simple and convenient. However,there are still shortcomings, that is, the interference of individual differences.

Conclusion

As the demand for soothing cosmetics has increased,various cosmetics that claim to have soothing effects have increased in number, so the evaluation method for soothing efficacy has been put forward with higher requirements. These days, the formation mechanism of sensitive skin is not fully understood,and comprehensive research is needed from various fields such as skin physiology, biochemistry,biomedicine and cosmetics science. This is also one of the important development directions in the research field of soothing cosmetics. In addition, the research and development of safe and effective cosmetic products will also be a topic of concern in the field of soothing cosmetics research.