Cai Yiwen,Liu Yuan,Song Chuang,Li Huiqiong

Guangzhou Huamiao Cosmetics Research Institute Co.,Ltd.,China

Abstract The aim of this study was to explore the mechanism of skin allergic reactions,and the role of skin allergic mediators in different types of allergic reactions.Further,we reviewed and classified anti-allergic safety evaluation models of cosmetics based on skin allergic media.In addition,the study explored in vitro experiments,cell experiments and animal experiments performed using anti-allergic safety evaluation model.The findings of this study provide information on the importance of anti-allergic safety evaluation models in cosmetics industry,and guides on selection of anti-allergic raw materials.Moreover,the findings of this study provide a basis for further research on development of mild cosmetics.

Key words cosmetics; allergy; anti-allergy safety evaluation model

Increase in number of cosmetics manufacturing industries increases demand for raw materials for production of cosmetics thus significantly increasing safety risks of cosmetics.Cosmetic Adverse Reaction Monitoring Network of the Ministry of Health of China reports that contact dermatitis caused by cosmetics allergy or irritation accounts for more than 90% of cosmetic skin diseases[1].Most cosmetic companies use plant extracts,active polypeptides and other active compounds to cosmetic products to prevent and relieve skin allergies.For example,chamomile is added to the product of French Soilya Cosmetics to improve the immune defense ability of the skin,whereas witch hazel extract is added to cosmetics produced in European and American countries to promote repair of skin allergic dermatitis[2].Notably,"anti-sensitive","safe without stimulation","mild without stimulation"and other terms are not indicated on cosmetic labels,however,the State Drug Administration is working on changing the regulatory policy.The Guidelines for Evaluation of Cosmetic Efficacy Claims published in September 2020 (draft for soliciting opinions)published efficacy evaluation principles of "claiming to be applicable to sensitive skin" and "claiming to be mild (no stimulation)",and recommends that enterprises should carry out evaluation tests following its guidance.

Clinical manifestations of cosmetic related skin allergic reactions include local skin itching,tingling,redness,desquamation,burning and tension[3].Clinical manifestations are broadly classified in to two types of type I and type IV allergic reactions,and the reaction principles of the two types are shown in Figure 1 and Figure 2.Type I allergic reaction refers to hypersensitivity reaction that occurs within a few minutes after the sensitized organism contacts the same antigen for a second time.It occurs quickly and subsides quickly,and mostly leads to physiological dysfunction in the organism and rarely causes tissue damage.Type IV anaphylaxis is an inflammatory reaction characterized by mononuclear cell infiltration and tissue cell damage caused by reaction between sensitized T cells and the corresponding antigen.Previous studies report that anti-allergic active compounds used against type I allergic reactions and type IV allergic reactions act mainly through inhibition of release of allergic mediators,or by antagonizing the action of allergic mediators to produce anti-allergic effect[4].

Figure 1.A flow chart showing Type I allergic reaction process

Figure 2.A folw chart for Type IV allergic reaction process

Several studies have explored anti-allergy activity of anti-allergy active substances throughin vitroexperiments,animal tests,cell tests and human tests.However,most studies only reviewed antiallergy determination methods and compared them based on efficacy.Only a few studies have report the mechanism of action of anti-allergy compounds against allergic reactions.Therefore,in this study the advances in anti-allergy safety evaluation models of cosmetics in recent years was reviewed mainly focusing on allergy mediators.The findings of this study provide a theoretical basis for screening of anti-allergy compounds and provide information on development of cosmetics with fewer allergic effects.

1 Histamine as a target for the assessment model

Histamine is a low molecular weight amine synthesized from L-histidine by histidine decarboxylase enzyme.The enzyme is ubiquitously expressed in various body cells,including mast cells,gastric parietal cells,and central nervous system.Histamine is implicated in type I allergic reaction,as it induces interactions between antigen and IgE antibody on mast cells.In addition,it causes expansion and increased permeability of capillary,promotes smooth muscle spasm and secretory activity and causes local congestion and edema.[5]Histamine can stimulate the skin directly causing skin allergy.The mechanism of action of histamine is shown in Figure 3.[6]

Biological effects of histamine are mediated by four types of histamine receptors,namely H1R,H2R,H3R,and H4R (Figure 3).[7]H1R and H4R receptors are implicated in mediation of skin allergies.H1R activation mediates several symptoms associated with type I anaphylaxis,including itching,erythema,and edema.Therefore,H1R antagonists are used for management of local and systemic allergic symptoms in type I anaphylaxis.[8]In addition,H4R receptors have been implicated in allergic diseases.In a previous study,co-administration of H1R and H4R antagonists inhibited occurrence of diarrhea and intestinal inflammation in experimental models presenting with peanut allergy.Furthermore,H4R knockout mice model of atopic dermatitis showed fewer dermatologic changes compared with wildtype mice.However,inhibitors of H4R have to be administered during sensitization and stimulation of the receptor to partially achieve similar results with those observed in H4R knockout mice.[9]

Figure 3.A diagram showing biological effect of histamine

Currently,several anti-allergic cosmetic agents against histamine are available in the market,such as Symcalmin? produced by SYMRISE in Germany,dipotassium glycyrrhizinate from Qinghai Lake Pharmaceutical,and Sens’flower? SD-SC from SEQENS in France.These agents have good inhibitory effect against histamine stimulation.Therefore,inhibition of histamine release directly confers anti-allergic effect.Several evaluation models based on biochemical reactions of histamine,cell experiments,[10]animal experiments,[11,12]and human experiments have been explored.

1.1 Hyaluronidase inhibition model

Studies report that release of histamine by mast cells increases hyaluronidase activity resulting in allergic reactions.[13]This implies that inhibition of histamine release by mast cell inhibits hyaluronidase activity.An in vitro inhibition model of hyaluronidase is used to determine sensitivity to histamine.Previous studies using hyaluronidase inhibition models showed that alkaloids,polyphenols,flavonoids(carvidin,luteolin,quercetin,kaamnesol and rutin)and terpenoids inhibit hyaluronidase activity.[14-16]Wang L used anin vitrohyaluronidase inhibition model to screen ten different extracts including cactus,purpus sl and sophora flavescens extracts.[17]The study reported that cactus,purpus sl and sophora flavescens extracts have good anti-allergic effects at similar concentrations.The anti-hyaluronidase effect of irisin extracted from the traditional Chinese medicine Radix Radix was explored by Liu C.[18]The study reported that pure irisin II effectively inhibits hyaluronidase activity.These findings imply that hyaluronidase inhibition rate can be used to evaluate anti-allergic effect of active compounds.

1.2 Cell experiments targeting histamine

Several cell experiments targeting histamine,including rat peritoneal mast cell degranulation model and xylene mouse ear swelling model have been established.Rat peritoneal mast cells degranulation model is used to simulate histamine release from mast cells.Peritoneal mast cells are harvested after intraperitoneal administration of a drug.The percentage of degranulated cells are then observed under a microscope after cell staining to characterize antisensitization effect of the drug.Rat peritoneal mast cell degranulation model was used by Ashwini[19]et al.,to study protective effect of Bresol? on mast cell degranulation and release of histamine from mast cells.In addition,the effect of Bresol? on mast cell degranulation induced by compound 48/80 was evaluated.The findings of the study showed that Bresol? inhibited histamine release from rat peritoneal mast cells.Furthermore,the herbal compound,Bresol? showed protective effect on mast cell degranulation.Mouse ear swelling model established by inducing xylene shows significantly high levels of histamine and other inflammatory mediators.These mediators stimulate local telangiectasia and aggravates inflammatory cell infiltration,causing acute exudative edema in the ear.Effects of different compatibility of Gentiana macrophylla and Gentiana macrophylla on experimental exudation and swelling in early inflammatory stage was explored using ear swelling experimental mice model.The findings of the study showed significant decrease in ear swelling in mice treated with G.macrophylla and G.macrophylla.[20]Therefore,animal experiments are the effective in evaluation of efficacy of antiallergy compounds.However,animal experiments are relatively complicated,and the European Union banned use of animals for evaluation of raw materials used in cosmetics since 2013,therefore animal models are not frequently used to study active ingredients in cosmetics.

1.3 Experimental model of histamine stimulation in human body

Experimental model of histamine human stimulation is established by application of histamine hydrochloride to the surface of human skin.Antiallergic ingredients are applied after the skin irritation produces redness,blisters and itching and alleviation of erythema and itching is observed.SYMRISE company used a specific test blade to drip histamine hydrochloride (10 mg/mL) on the skin test site.A test product (Symcalmin? 2 mg/cm2)was applied after the skin developed blisters and itching.Itching,redness and blister area of the test site were measured 30,60,90,120,and 150 minutes after the test product was applied.The results showed that Symcalmin? significantly reduced the adverse reactions caused by histamine,such as itching,redness and blisters,in a dose-dependent manner.Differences among individuals may lead to differences results because each individual has different skin sensitivity,therefore,contributed use of the product is determined by the effect on the skin.Therefore,human trials are required for evaluation of the efficacy of active compounds.[21]

2 Evaluation model of arachidonic acid metabolism pathway as target

Arachidonic acid (AA) mainly occurs in animal tissues.The molecular structure of AA is shown in Figure 4.It accounts for 40% of molecules in human brain and nerve tissue,and up to 70% in nerve endings,whereas phospholipids contain 1% of AA.AA metabolic pathway is an important target in type I allergic reaction.The metabolic pathway of AA is shown in Figure 1.Phospholipids in membrane are degraded by phospholipase to generate AA,which is then metabolized to thromboxane,prostaglandin PGE2,or to leukotriene,platelet activator,bradykinin and other allergy mediators.[22]Leukotriene A4 hydrolase (LTA4H) is a key enzyme in AA metabolic pathways.Hydrolysis by LTA4H produces inflammatory factor leukotriene B4(LTB4).LTB4 is implicated in a variety of diseases such as inflammation,therefore inhibition of LTB4 activity in cells or reduction of LTB4 synthesis is applied in treatment of a variety of diseases such as inflammation.The targeting of LTB4 can be used to design anti-inflammatory drugs.[23]

Figure 4.Molecular structure of arachidonic acid

2.1 Model of cell light damage induced by UVB

UVB irradiation causes acute inflammation of the skin.In addition,epidermal keratinocytes directly stimulated by UVB releases several inflammatory mediators,resulting in telangiectasia,and erythema of the skin.[24]COX-2 plays an important role in the occurrence of inflammation induced by UVB.COX-2,a subtype of Cyclooxygenase,is a key ratelimiting enzyme in the process of degradation of AA to produce various endogenous prostaglandins(PGE2).PGE2 stimulates synthesis and accumulation of inflammatory factors in damaged sites,[25]promoting inflammatory response and tissue damage.Therefore,COX-2 is a valuable indicator for studying inflammatory response of skin.The UVB induced cell photoinjury model is used to culture epidermal keratinocytes.The model is established by irradiating epidermal keratinocytes with different doses of UVB,and the content of various allergic mediators in the UVB damaged cells are detected.The UVB induced cell light damage model established by Zhou[26]showed that polydatin reduced expression of apoptotic genes and inflammatory genes,such as expression of p53,caspase-3,and baxmRNA.In addition,expression of inflammatory genes such as IL-6,IL-18,was inhibited and expression of matrix metalloproteinase MMP-1 was inhibited.IRB Company pretreated human epidermal keratinocytes with different concentrations of GPI for 24 h,and then exposed the cells to UVB (25 MJ/cm2)irradiation to determined secretion of levels of PGE2.The findings showed that GPI significantly inhibited secretion of PGE2 induced by UVB in epidermal keratinocytes and alleviated inflammation.In a previous study,He explored the effect of cornein-3-glucose in ROS/COX-2 pathway of HaCaT cells damaged by UVB.[27]The findings showed that UVB promotes phosphorylation of ERK,p38,JNK and Akt,regulates expression of COX-2,and reduces synthesis of inflammatory factor prostaglandin(PGE2),thus reducing erythema on skin.

2.2 AA induced ear swelling model in mice

AA-induced mouse ear swelling model is widely used in testing anti-allergic effect of compounds.Ear swelling is mediated by leukotriene and other fat oxidases,Prostaglandin (PGE2) significantly promotes ear swelling in mice in presence of leukotriene.[28]Li et al.,explored anti-sensitivity effect of (+)-2-(1-hydroxyl-4-oxocyclohexyl)ethyl caffeate,JH2[29]using AA-induced mouse ear swelling model.The findings showed that JH2 significantly reduces the degree of AA-induced ear swelling and reduces amount of tissue protein exudation in mice.In addition,JH2 reduced production of leukotrienes B4 in inflamed ear tissues of mice.Bhat[30]et al.carried out animal experiments and reported that resveratrol inhibits COX-1 and reduces PGE2 synthesis,implying that resveratrol is a good anti-inflammatory compound.A mouse ear swelling model induced by arachidonic acid administration was used to explore inhibitory effect of THCA354 compound on acute inflammatory response.[31]The findings showed that THCA354 significantly inhibited ear swelling in mice in both the experimental group and the positive control group.

2.3 A platelet aggregation model induced by AA

AA-induced platelet activation factor (PAF),a platelet aggregation activator promotes thrombosis and induces inflammatory response.[32]Platelet aggregation model is established through PAFmediated platelet aggregation.Zang explored antiinflammatory effects of hydroxyl safflower yellow A and Myricetin.[33]The active parts of safflower that promotes blood circulation and removes blood stasis are anti-PAF active components.These compounds inhibits specific binding of PAF with rabbit platelet receptor,and significantly inhibits PAF-induced platelet adhesion.

3 Test model with IgE as target

IgE is a 190 kD immunoglobulin produced by B lymphocytes and which plays an important role in allergic reactions.Its activity is related to the protein network,and its structure is shown in Figure 5.[34]There are two types of IgE receptors including low-affinity IgE receptors expressed on B cells and other hematopoietic cells (FcεRII; CD23) and high affinity IgE receptors(FcεRI).FcεRI is expressed as a tetramer (αβγ2) on mast cells and basophil granulocytes,and as a trimer (αγ2) on antigen presenting cells.The level of FcεRI expression in human basophil cells is positively correlated with serum IgE levels.IgE binding on basophil cells inhibits the receptor on the cell surface whereas FcεRI subunit has no known enzymatic activity,however,it involved in signal transduction through associated cytoplasmic tyrosine kinases.[35]Previous studies report that IgE and mast cells are key drivers of long-term pathophysiological changes and tissue remodeling associated with chronic allergic inflammation in asthma and other conditions.Inflammatory activity is mediated through IgE dependent regulation of mast cell function,effects independent of mast cell IgE,and effects of mast cell roles that are not directly mediated through IgE.[36]Therefore,several studies are exploring design of drugs that target IgE signaling pathway,block binding of IgE and its high affinity receptor (FC) and downstream signal transmission for development of novel therapies for allergic diseases.

Figure 5.Structure for immunoglobulin E (IgE)

FcεRI activation of mast cells has been explored through skin test or determination of allergenspecific IgE in serum.[37]The study reported that FcεRI activation is implicated in pathogenesis of allergic conditions,such as skin erythema,edema and itching.A study recruited a total of 1 100 patients with allergic skin diseases and their serum total IgE value was determined.[38]The study reports that 53%of the patients with allergic skin diseases showed significantly high serum total IgE concentrations.Patients presenting with eczema and contact dermatitis showed that serum IgE plays an important role in occurrence and development of allergic skin diseases.

A previous study explored the activity of plants used by the Golden flower tea group against IgE mediated type I allergic reaction through determination of IgE levels.[39]The study findings showed that plants used by the golden flower tea group significantly reduced levels of IgE and LT in serum,amount of EOS in whole blood,and the inflammatory area of rat lung tissue.Qinghai Lake Pharmaceutical treated mouse macrophages stimulated by histamine with glycyrrhizin and glycyrrhetinic acid.The study reported that inhibitory effect of glycyrrhizin at 1 mg/mL was 83.4% through determination of IgE levels,which was significantly higher compared with that of glycyrrhetinic acid.

4 Evaluation model with cytokines as targets

Cytokines are peptides or glycoproteins secreted by immune cells and section is regulated by the NFκB signaling pathway.Cytokines bind to specific receptors on target cells.[40]which are implicated in type IV allergic reactions (Figure 2).The main classes of cytokine include tumor necrosis factor:TNF-α,TNF-β,interleukin:IL-1α,IL-6,IL-8 and colony stimulating factor:G-CSF,GM-CSF.

4.1 A keratinocyte model developed through stimulation with TNF-α

Keratinocytes produce high amounts of cytokines,and acts as target cells for many cytokines,thus playing an important role in skin immune response.Serial dilutions of TNF-α with a maximum concentration of 10 ng/mL to keratinocyte cell culture medium treated with 20 μM daphnetin[41].Analysis showed inhibition of increase in inflammatory factor expression induced by TNF-α stimulation of keratinocytes.A study using Enzymelinked immunosorbent assay (ELISA) showed that ecdysterone inhibits production of inflammatory cytokines in keratinocytes after TNF-α stimulation.[42]

4.2 Macrophage model of RAW264.7 developed through lipopolysaccharide (LSP)administration

Lipopolysaccharide (LSP) is a harmful inflammatory factor composed of hydrophilic O-specific antigen polysaccharide,lipid A,a core polysaccharide and a side chain.Lipopolysaccharide A comprises glucosamine,phosphate and a fatty acid moiety,which are responsible for the biological activity of LSP.The mechanism of LSP induced inflammatory response in RAW264.7 is through formation of LSP-LBP-SCD14 complex which acts on TLR4.In addition,this complex activates NFκB and MAPKs signaling pathways,induces mRNA expression of iNOS and COX-2 in RAW264.7 macrophages,promotes transcription of inflammatory genes such as iNOS and COX-2,and induces release of pro-inflammatory cytokines such as TNF-α,IL-6 and IL-1β.[43]A study using LPS-induced RAW264.7 macrophage model showed that the levels of TNF-α,IL-6 and IL-1β were significantly reduced after pretreatment with liensinine.[44]This finding shows that liensinine has anti-inflammatory effect.A different study screened and isolated the active compounds of odontoglum and tested them using the macrophage model stimulated by LPS.Analysis showed that active compounds extracted form odontoglum inhibited release of TNF-α in RAW264.7.[45]

4.3 A ear swelling mouse model established through treatment with TPA

TPA induces keratinocytes of mouse ear tissue to produce TNF-α,IL-1β and other inflammatory cytokines.TNF-α promotes secretion of secondary inflammatory cytokines,inducing inflammatory response.In addition,TNF-α stimulates phospholipase to induce release of AA,which is metabolized into PGE2 and LTB4,promotes release of histamine and 5-hydroxytryptamine,and aggravates inflammation.A previous study reports total saponins of Panax pengbiannotoginseng inhibit secretion of inflammatory mediators NO,PGE2 and TNF-α.[46]In addition,total saponins significantly reduced edema,capillary dilatation and inflammatory cell infiltration in a mouse ear swelling model induced by TPA.The auricle swelling model induced by TPA was used to evaluate the anti-inflammatory activity and mechanism of active metabolites from active yeast cells.[47]The findings showed that the metabolites from active yeast cells significantly reduced thickness of auricle cuticle induced by TPA,inhibited expression of inflammatory factor COX 2 and blocked activation of NF-B pathway,with approximately 54.46%.inhibition rate.

5 Capsaicin receptor (TRPV1) pathway as a target evaluation model

Capsaicin receptor (TRPV1),a non-selective cation channel is ubiquitously expressed in mammalian sensory nerve fibers.[48]Activation of TRPV1 induces flow of Ca2+,Mg2+,Na+and other cations into the cell,however,it has relative selective specificity for Ca2+and Mg2+.Activation of TRPV1 increases intracellular cation concentration resulting in physiological and pathological changes(Figure 6).[49]TRPV1 can be directly or indirectly activated by a variety of neuroinflammatory mediators and endogenous inflammatory mediators such as bradykinin,substance P,prostaglandin,nerve growth factor and calcitonin gene-related peptide (CGRP).[50]CGRP is the first neuropeptide to be developed through gene recombination and biosynthesis,and comprises a total of 37 amino acids.TRPV1 is a key receptor for regulating synthesis and release of CGRP.

Figure 6.TRPV1 structure

5.1 A mouse ear oedema model established by capsaicin treatment

The mouse ear edema model is established by administration of a certain dose into the ear of mice.Swelling then through mediations of neuropeptides such as calcitonin gene-related peptide,substance P,neurokinin A and vasoactive intestinal peptide.A previous study evaluated the local anti-inflammatory effect of pericarp extract using the mouse ear edema model which showed that the extract reduced ear edema in mice.[51]

5.2 Experimental model of capsaicin stimulation in humans

Capsaicin human stimulation model is established by mixing capsaicin and samples to be tested which are then applied to human skin surface to induce skin prickling,burning and subsided conditions.Symrise applied an oil-in-water emulsion containing 31.6 mg /kg capsaicin and 1% of the active substance to the nasolabial crevices of volunteers.The study showed that 1% of the active substance significantly reduced the tingling and burning feeling caused by capsaicin compared with the sample containing only capsaicin.

6 Other measurement models

In addition,measurement of skin physiological function indexes such as TEWL,water content,pH value and Lab value before and after use of products is important for evaluation and verification of the actual effects of anti-allergic agents and cosmetics on human bodies.[52]

7 Conclusion

Abuse of various industrial additives due to deterioration of the ecological environment has increased skin adverse reactions,such as low skin tolerance,vulnerability to a variety of physical and chemical factors to stimulate allergies,resulting in tingling,burning,tension,itching and other manifestations.It is important to add anti-allergenic active ingredients to cosmetics to alleviate adverse effects of cosmetic raw material compounds and to reduce irritation to the skin.Therefore,studies should explore the mechanism of allergic reaction and design appropriate anti-allergy safety assessment methods for screening of anti-allergy compounds used in cosmetics.