Establishment And Application Of Evaluation Method For Anti-UV Injury Activity Based On Metabonomics

Ultraviolet radiation can cause a series of health damage.The anti-ultraviolet active substances can effectively prevent,reduce and repair the damage caused by Ultraviolet radiation.At present,the evaluation methods of anti-ultraviolet activity mainly include physical chemistry methods and biological methods.The former is simple and intuitive,but it cannot express the changes of pathophysiology and effect level induced by radiation.The latter assesses histological,morphological changes and the effect level caused by radiation in cells,tissues,animal and human body.And yet,it is difficult to reflect the overall changes in the body with limited observation index.Metabonomics studies the relative relationship between the metabolites and the changes of physiology and pathology by analyzing all small molecule metabolites.Anti-ultraviolet activity of bioactive substances is evaluated comprehensively through the changes in the metabolites.In this paper,the skin biomarkers of UV-injured mice were indentified through using the metabolomics and pattern recognition,besides a novel method for evaluating the activity of anti-ultraviolet injury was established and applicated in evaluating the four kinds of peptides.Objective: In order to develop a method based on metabolomics to evaluate the activity of anti-ultraviolet injury via combining with chemometrics,,the acute UV damage model of Kunming mice skin was establish and the difference of endogenous metabolites between the control group and the model group was analyzed.Then the anti-UV activity of peptides was evaluated by the established methods.Method:(1)TheUPLC-DAD conditions were optimized by adjusting the parameters of flow phase composition,detection wavelength and velocity.(2)The SPF male Kunming mice were divided into control and model groups.The acute ultraviolet injury model was established by exposing to UV radiation for 12 hours and verified by observing the pathologic changes of frozen section.The mice skin tissue was taken and analyzed after series pretreatment.Data were analyzed using pattern recognition to establish a statistical model for ultraviolet damage,then the biomarkers were found and identified.The metabolic pathways and mechanisms of biomarkers may be explained by retrieving literature and KEGG database.(3)The SPF male Kunming mice were divided into the control group,model group and intervention group,the latter two were exposed to UV irradiation for 12 hours to construct the anti-ultraviolet damage model.A statistical model of anti-ultraviolet damage was established using PCA and OPLS-DA,and the evaluation method of anti-ultraviolet activity was developed by Y prediction method,the anti-ultraviolet activity of the target polypeptide was evaluated subsequently.Results:(1)The optimal chromatographic conditions were: a flow rate was 0.25 m L/min;column temperature was 30°C;chromatographic column was performed on an Agilent SB-Aq RRHD column(100 mm x 2.1 mm,1.8?m);the mobile phase was 0.2% phosphoric acid and acetonitrile;the solvent gradient elution was 0.5 % acetonitrile(0-2 min),0.5-25% acetonitrile(2-6 min),25-95% acetonitrile(6 – 8.5 min),95% acetonitrile(8.5-10 min);the wavelength was 254 nm.(2)The model of ultraviolet acute injury was established successfully through pathological examination of frozen section.Multivariate statistical analysis shows a significant difference in the model group and the control group and there are 18 peaks with the VIP > 1.Three potential biomarkers were identified: uracil nucleoside,hypoxanthine nucleoside and 3-nitrotyrosine.(3)Based on metabolomics analysis and pattern recognition method,a method for evaluating the activity of anti-ultraviolet injury was developed.Using skin care product,sunscreen,VC and homosalate as the positive control groups,four kinds of peptide were evaluated.The results shows that the order of anti-ultraviolet activity was with the skin care product > peptide 1 > sunscreen > polypeptide 2;homosalate > polypeptide 3>VC> polypeptide 4.Conclusion: Combined with metabolomics and pattern recognition,a novel method was established to evaluate the anti-ultraviolet activity,besides the anti-ultraviolet activity of the polypeptide was evaluated by the developed method.It has a high degree of fitting and prediction,and there is no over-fitting,which can be used for the qualitative and relative quantitative evaluation of anti-ultraviolet active.