Guo Shentao,Xu Wenfeng,Zhang Yuling

Guangdong Youzhi Testing Technology Co.,Ltd.,China;

Long Yan

Guangdong Bawei Biotechnology Co.,Ltd.,China

Abstract In order to search an appropriate evaluation method for the soothing effect of cosmetics,the LPSinduced macrophage model,SDS-stimulated zebrafish embryos model and the clinical assessment were used.The results showed that the concentration of NO and IL-6 was significantly reduced in LPS-induced RAW264.7 cells when applied to two essences.The inhibition rate of NO-release was 85.87% and 84.57%,while IL-6 was 44.18% and 38.76%,respectively.Furthermore,the spontaneous rotating of zebrafish embryos was significantly decreased to 51.85% and 76.54% in SDS-stimulated zebrafish embryos model.In the 28 days of clinical assessment,the concentration of hydration was significantly increased to 55.56% and 39.61%,while the erythrodesis was significantly decreased to 4.75% and 6.68%.This indicates that the three evaluation methods of soothing efficacy in this research were effective,and can be used in the evaluation of cosmetics.

Key words cosmetics;soothing efficacy;efficacy

Skin irritation is mainly caused by the increase of capillary permeability and secretion of mucosal glands after the destruction of skin barrier,which often accompanied by skin swelling,itching,plaque and other phenomena[1].Although there were many ways to induce skin irritation,irritating substances first damaged the skin surface,resulting in abnormal cuticle structural protein,decreased lipid,increased permeability,increased water loss through the epidermis and decreased hydration,leading to abnormal skin barrier function[2].With the destruction of the skin barrier,various stimulants induced the release of inflammatory factors such as IL-6,IL-8,and TNF-α from keratinocytes,which triggered the skin immune inflammatory response[3,4].

The soothing efficacy of cosmetics refers to the improvement of skin stimulation after using cosmetics[5].Inflammatory response due to skin irritation was an important physiological response in the immune system[6],which played an important role in skin stimulation.It can be effective to improve the stimulation of skin by reducing the inflammatory reaction.There were currently no national or industry standards for the evaluation methods of soothing efficacy,and the testing methods mainly refered to group standards such as clinical assessment or cytoinflammatory factor detection[7-9].However,the consistency between the evaluation methods of laboratory and clinical assessment remained unclear.In order to evaluate the feasibility of different methods,LPS -induced RAW264.7 Cells,SDSstimulated Zebrafish embryos and clinical assessment were used to evaluate the two essences,so as to provide reference for the evaluation and application of soothing efficacy.

1 Materials

Mouse mononuclear macrophages,RAW264.7 cell line,was obtained from China center for type culture collection (CCTCC).Wild-type adult zebrafish(AB line),was purchased from China Zebrafish Resource Center(CZRC).DMEM,fetal bovine serum,Trypsin-EDTA,Gibco,USA.Lipopolysaccharide(LPS),MTT,DMSO,Griess reagents,were obtained from Sigma-Aldrich,USA.Sodium dodecyl sulfate(SDS),were purchased from Macklin,China.IL-6 Elisa kit,was procured from Wuhan Huamei,China.Essence 3# and 4#,were obtained from Guangdong Bawei Biotechnology Co.,Ltd.

2 Method

2.1 Cell cultrue

Raw264.7 cells were routinely maintained in DMEM,containing 10% heat-inactivated FBS,at 37 ℃in 5% CO2incubator,and experiments were taken after 24～48 h incubation in 96-well plates.

2.2 Zebrafish husbandry and embryos collection

Adult zebrafish were kept in a recirculating aquarium system with controlled conditions for a week at (28±1) ℃ on a 14 h light/10 h dark photoperiod cycle.Male and female adult zebrafish at the ratio of 1:1 were let to a collection container on the night before experiment,embryos were obtained from natural spawning,which was induced in the next morning,by turning on the light,and fertilized eggs were collected 1 h post light onset.Embryos were washed twice with embryo media,cultivated at (28±1) ℃ for 24 h,good healthy embryos(24 phf)were picked to experiment.

2.3 Soothing evaluation by LPS -induced RAW264.7 Cells

Raw264.7 cells (5 × 104cells/well in 96-well for 24 h incubation) were stimulated with LPS (1.25 μg/mL) in the presence or absence of essences for 24 h simultaneously.Nitric oxide production was measured by Griess assay,and the levels of IL-6 in the cultured medium of Raw264.7 were measured using an IL-6 ELISA kit,performed according to operating protocol of the kit.Dexamethasone sodium phosphate was used as positive control(PC),the medium with or without LPS were used as negative controls (NC) and blank control (BC).For griess assay,50 μL of culture medium was incubated with 50 μL Griess reagent for 10 min in darkness.The production of NO was determined by measuring optical density at 540 nm.NO production by individual LPS stimulation was designated as 100% for each experiment.NO relative content,NO inhibition and IL-6 inhibition rate were calculated as follows:

NO relative production=Asample/ANC×100%

NO inhibition activity=(ANC-Asample/ANC×100%

IL-6 inhibition rate=(1-T/ C) ×100%

Where ANC and Asample were the absorbance of NC and Sample,respectively.T is the average of IL-6 in sample group,and C is the negative control.

2.4 Soothing evaluation by SDS -stimulated zebrafish embryos

Zebrafish embryos at 24 hpf were selected and arrayed in 96 well-plate with 5 embryos per well.Samples were diluted to a nontoxic concentration and added to the 96-well plate with 100 μL per well.After incubated at (28±1) ℃ for 60 min,the zebrafish embryos in 96-well plate were stimulated with 500 μmol/L of SDS,dilution with and without SDS were set as negative control (NC)and blank control (BC).For 15 min incubation,the spontaneous rotating of zebrafish embryos were recorded by video.

2.5 Clinical assessment

To determine the clinical efficacy of soothing in each essence,we studied no less 30 healthy volunteers,who was sensitive to the lactic acid tingling.Subjects applied the essences to the full face every day for 4 weeks and then,face images were taken every 2 weeks for erythrodesis,and the moisture content of skin were determined.

2.6 Statistical analysis of the data

A t-test was used to compare statistical differences between experimental and control groups for cell and zebrafish experiments.For clinical assessment,A t-test was used when the data was normality according to SPSS,or the Wilcoxon-test was used.Ap<0.05 was considered statistically significant.

3 Results and discussion

3.1 Soothing evaluation by LPS -induced RAW264.7 Cells

Using LPS to induce acute inflammation in Raw264.7 cells,we found that the NO and IL-6 production were significant increased (P<0.0001)compared to BC,and the PC dramatically reduced LPS-induced NO and IL-6 production,with the inhibition activity 22.29% and 23.18%,that indicated the soothing evaluation of cell model was succeed.The inhibitory rate of essence 3#and essence 4# was 85.87% and 84.57% in NO production,while 44.18% and 38.76% in IL-6 (Table 1,Table 2),the result showed that both of the two samples had a soothing efficacy.

Table 1. Relative content of NO in the LPS -induced RAW264.7 Cells

Table 2. Content of IL-6 in the LPS -induced RAW264.7 Cells

Table 3.Test result of SDS-stimulated Zebrafish embryos

3.2 Determination of spontaneous rotating by SDS-stimulated zebrafish embryos

Using SDS to stimulate irritation in zebrafish embryos,we found that the spontaneous rotating were significant increased (P<0.05) compared to BC,indicated the soothing evaluation of zebrafish embryos model was succeed.The inhibitory rate of essence 3#and essence 4# was 51.85% and 76.54% in spontaneous rotating,showed that both of the two samples could relieve the irritation of zebrafish embryos.

3.3 Clinical assessment of soothing efficacy

60 healthy volunteers were tested in this experiment,each group for 30 volunteers.For essence 3#,after 28 days’ treatment continuously,the hydration increased to 55.56%,and the erythrodesis decreased by 4.75%,which was significant compared with basal values (P<0.01).Likewise,the results obtained for essence 4#revealed that 4weeks after treatment,the hydration increased to 39.61%,and the erythrodesis decreased by 6.68%,when comparing with basal values,with significant (p<0.05) .

Figure 1. Clinical assessment of soothing efficacy treated by Essence 3#

Figure 2 Clinical assessment of soothing efficacy treated by Essence 4#

4 Conclusion

In this study,LPS -induced RAW264.7 Cells,SDS-stimulated Zebrafish embryos and clinical assessment were used to evaluate the two essences.It can be seen that the three methods were feasible and can be used as one of the methods for the soothing efficacy evaluation of cosmetics,according to the design and result analysis of experiments.The application of methods can provide scientific support to screen ingredients,formula design,efficacy verification and efficacy claim of cosmetics.

Both cell and zebrafish model are laboratory methods,which were good replacement models for human tests,and the results can be used for efficacy claim directly.In the zebrafish model,the spontaneous rotating of each embryo were significant different,influenced by the quality of zebrafish embryos.Moreover,the samples directly added to the embryo culture medium can be taken orally and got more toxicity,the mechanism was different from the percutaneous absorption of the skin,which had great limitations.The cell method also had the limitations of mechanism of action and sample dosage form,but it can be economical,fast,controllable,standardized and high repeatability,which can effectively avoid the large result deviation caused by individual differences of human body,and had a more extensive application prospect.The results showed that the three different evaluated methods were effective for the same sample,and they all can be used as one of the methods of soothing efficacy evaluation.In practical application,when the soothing efficacy is ineffective through one of the evaluated method,other methods can be reasonably applicated for evaluation supplementary,according to the mechanism of action and the usage,so as to avoid the wrong results caused due to the limitations of the method.