Spirulina-derived ACE Inhibitory Peptides For Inflammageing Regulation And Its Safety Evaluation In Cosmetics

Local renin-angiotensin system（RAS）is also present in skin tissues and involved in the regulation of various physiological and pathological activities of skin such as wound healing,inflammation,psoriasis and aging.Angiotensin-converting enzyme（ACE）plays a central role in the regulation of the RAS.Some ACE inhibitors have been proved to improve scarring and skin photoaging.However,these ACE inhibitory drug have more serious side-effects,so the development of natural ACE inhibitory peptides from food-derived proteins has provided a new direction for the application of ACE inhibitors.In this study,the ACE inhibitory peptide from Spirulina was prepared by protease using Spirulina protein.The peptidomic and computational simulation methods were combined together to screen ACE inhibitory peptide.L929 cells and proteomics were used to explore thier biological activity.The safety and stability of the ACE inhibitory peptides were also evaluated.The major research content are as follows:（1）The best protease for Spirulina was selected among alkaline protease,neutral protease,pepsin and trypsin based on the degree of hydrolysis,ACE inhibition rate and DPPH radical scavenging rate.The results showed that the hydrolyzed product by alkaline protease had the highest degree of hydrolysis,ACE inhibition rate and DPPH radical scavenging rate.The peptides from alkaline protease hydrolysate were analysed by LC-MS/MS.A total of 782peptides were identified.Two new ACE inhibitory peptides,HIIARPH(IC₅₀=461.5μmol/L)and LRLKE(IC₅₀=155.8μmol/L)were screened using pharmacophore modeling and molecular docking.The Lineweaver-Burk double inverse plotting method indicated that the inhibitory mode of HIIARPH and LRLKE were non-competitive inhibitory mode.（2）Western Blot showed that Ha Ca T,NIH 3T3,and L929 cells can express ACE.Skin fibroblasts L929 had the highest expression and were selected for the further study.The cytotoxicity of HIIARPH and LRLKE was examined by MTT,and both inhibitory peptides were not significantly toxic to L929 cells at a concentration of 1.0 mmol/L.L929 cells were stimulated with LPS to establish a inflammageing cell model.The m RNA expression of ACE and AT₁R were measured by RT-PCR.The protein expression of ACE and Ang II were measured by Western blot.Ang II in culture supernatant was measured by ELISA.The results showed that LPS activated the RAS,increased the m RNA and protein expression of ACE/Ang II/AT₁R pathway.Finally,the concentration of LPS were 1,000 ng/m L.L929 cells were treated with different concentrations of HIIARPH and LRLKE with 1,000 ng/m L LPS and intracellular SOD and CAT enzyme activities were measured.The results showed that both HIIARPH and LRLKE increased the enzymatic activities of the antioxidant enzymes SOD and CAT.Moreover,LRLKE showed better antioxidant activity than HIIARPH.ELISA was used to detect the secretion of senescence-associated secretory phenotype（SASP）IL-6,and it was found that HIIARPH and LRLKE could reduce the secretion of IL-6.HIIARPH（1.0 mmol/L）of and LRLKE（1.0 mmol/L）were used to evaluate the effect of HIIARPH and LRLKE on RAS.The ACE activity in the culture supernatant was measured by ELISA.The m RNA expression of ACE,AT₁R and AT₂R was measured by RT-PCR.The protein expression of ACE and AT₁R was measured by Western blot.The results showed that both HIIARPH and LRLKE were able to inhibit ACE activity in supernatants and inhibit the ACE/Ang II/AT₁R pathway in RAS.In addition,LRLKE was able to further activate AT₂R.（3）The differential expression of proteins involved in the regulation of LRLKE in L929cells induced by LPS was further analysed by proteomics.The results verified that LRLKE was involved in the regulation of various signalling pathways such as cell senescence,p53,NF-κB,viral infection,ferroptosis and autophagy in L929.The differential proteins in cellular senescence and apoptosis signalling pathways were also validated by Western Blot and ELISA based on protein enrichment analysis.The results proved that the proteomic data were reliable and valuable for reference and LRLKE could regulate cellular inflammageing and apoptosis via ACE/AT₁R/NF-κB/IL-6.LRLKE could regulate cellular apoptosis via ACE/AT₁R/Bcl-2/Bax and ACE/AT₁R/Bid/Bax.（4）The safety of HIIARPH（1.0 mmol/L）and LRLKE（1.0 mmol/L）was evaluated.Direct polypeptide reaction assay（DPRA）was used to evalutated the sensitization of two ACE inhibitory peptides.The chick embryo allantoic membrane assay（HET-CAM）and mouse erythrocyte hemolysis were used to evalutated the irritation of two ACE inhibitory peptides.The Ames assay was used to evaluated the mutagenicity of two ACE inhibitory peptides.The patch texperiment was used to test the human safety.The results showed that HIIARPH（1.0mmol/L）and LRLKE（1.0 mmol/L）were not irritating,allergenic and mutagenic and are safe to human.（5）The temperature,p H and storage stability of HIIARPH and LRLKE were tested further.The results showed that LRLKE was more stable and less affected by changes in temperature and p H.In contrast,HIIARPH showed a significant decrease in ACE inhibitory activity when temperature was more than 50°C and p H was less than 6.In addition,storaged at0°C and 25°C for 16 days had no significant effect on the inhibitory activity of the two ACE inhibitory peptides.