The Repairing Effects And Application Of Linseed Orbitides On Skin Photodamage

Linseed orbitides（LOs）are homocyclic peptides connected by peptide bonds.Studies have shown that LOs exhibited kinds of biological activities,including antioxidant,anti-inflammatory,immunosuppressive,anti-malaria,anti-cancer,chelated metal ions and so on.Therefore,LOs have great application potential in food,cosmetics and pharmaceuticals.However,the application of LOs was limited by the imperfection of purification process.In order to promote the development and application of LOs,this paper used methanol-K₂HPO₄aqueous two-phase system（ATPS）to effectively improve the purity of LOs in purified products,and explored the role of LOs in the repair of skin photodamage to promote the application.The main research contents are as follows:Firstly,the purification process of LOs was studied by using methanol-K₂HPO₄ aqueous two-phase system.Under the conditions of salt content of 14%,methanol content of 44%,solid-liquid ratio of 1:30 and p H 8.7,the phase ratio of ATPS was 4.91±0.11,and the extraction rate and partitioning coefficient of LOs were 79.02±1.08%and 0.77±0.03,respectively.Then,the methanol phase was separated.And the purified products were obtained by the steps of desolvation,washing and lyophilization.And 13 kinds of LOs were identified from them,and the purity reached 76.36%.Secondly,according to the REDOX properties,LOs were classified into oxidized linseed orbitides（OLOs）and reduced linseed orbitides（RLOs）by preparation chromatography,and their physical and chemical indexes were determined.The results showed that RLOs was mainly composed of LOB,LOL,LO-A+P and LO-M+O with the contents of 16.68%,6.19%,68.36%and 6.23%,respectively,and the purity was 97.08%.OLOs included LOF,LOG,LOC,LOE,LO-D+J and LON,with the content of 4.23%,22.84%,12.02%,19.72%,8.34%and4.23%,respectively,and the purity was 80.50%.In vitro free radical scavenging experiments showed that RLOs had a stronger ability to remove·O^2-,H₂O₂ and·OH from OLOs,with the highest scavenging rates of 45.61±3.14%,57.10±0.95%and 11.07±1.45%,respectively.Then,the repair effects of RLOs and OLOs on skin photodamage were compared by using zebrafish model.The results show that RLOs is more suitable for repairing skin photodamage.After 100μg/m L OLOs and RLOs treatment,the area of Ultraviolet B（UVB）irradiated zebrafish caudal fin was restored from 65.74±4.74%to 67.24±7.37%and 98.16±6.73%,respectively.Similarly,the ROS accumulation induced by UVB irradiation recovered from 232.38±42.44%to 205.38±26.74%and 106.68±18.86%,respectively.Further studies showed that RLOs could alleviate the UVB induced oxidative stress by improving the activity of endogenous antioxidant enzymes and regulating the homeostasis of intracellular calcium ions to inhibit the oxidation of protein and lipid,and promote the repair of photodamage in zebrafish.Finally,focusing on the problem of poor water solubility of RLOs,a solution using co-solvent solubilization was proposed.And the RLOs essence product was initially developed,and its photodamage repair effect was evaluated.The results showed that arginine had a significant effect on improving the water solubility of RLOs.When the concentration of arginine was 100 mg/m L and the stirring time was 20 h,the solubility of RLOs in water increased from 0.78±0.01 mg/m L to 8.75±0.12 mg/m L,which could meet the requirements of the product.Then,the effect of RLOs essence on repairing photodamage was evaluated,and the results showed that when the content of RLOs in the essence reached 0.4%,the skin redness and melanin pigmentation of rats caused by UVB could be significantly relieved.In conclusion,methanol-K₂HPO₄ aqueous two-phase purification strategy was used to effectively improve the purification process of LOs,and the method was efficient,simple and easy to operate.Secondly,RLOs had a better repair effect on skin photodamage,and its mechanism was described.Finally,the water solubility of RLOs was improved by co-solvent,and the preliminary development of RLOs essence could effectively inhibit the aggravation of photodamage at the initial stage,and had a good repair effect,which provided a reference for the application of LOs.