Toxicological Safety Evaluation Of Cyclopeptide E In Flaxseed Oil

Cyclopeptide E（CLE）is a special hydrophobic cyclic peptide in flaxseed oil,which is composed of eight to nine amino acids.Studies have shown that cyclic peptides have anti-inflammatory,anti-cancer,anti-oxidation and other biological activities,so cyclic peptides have potential application value in the field of medicine.At the same time,cyclopeptide E is also the main source of bitterness in linseed oil.At present,most of the research on the bitterness of linseed oil stays in the identification and debittering of bitterness.However,whether the bitterness in flaxseed oil is toxic to the human body has not been reported,in order to scientifically evaluate its safety,this study used mouse acute toxicity test,subacute toxicity test,mouse bone marrow cell micronucleus test and mouse sperm deformity test to evaluate the safety of cyclopeptide E in flaxseed oil.The safe use dose of cyclopeptide E was explored to provide a reference for the subsequent development and utilization of cyclopeptide E.The specific research contents and results are as follows:（1）The cyclic peptides in flaxseed oil were extracted,and the qualitative analysis of cyclic peptides was carried out by UPLC-Q-TOF/MS.According to the mass-to-charge ratio and secondary spectrum verification,it was concluded that flaxseed oil contained cyclic peptides.The external standard method was used for quantification.After repeated extraction of methanol solvent for 3 times,the recovery rate of standard addition could reach 96.62%.The initial content of cyclopeptide E in linseed oil used in this experiment was 702.01 mg/kg.Subsequently,methanol was repeatedly extracted and flaxseed oil was used as a solvent for continuous reconstitution,and finally the highest extraction concentration of cyclopeptide E was 5000 mg/kg,and subsequent experiments were carried out on the basis of this concentration.（2）In the acute toxicity test,a dose of 5000 mg/kg·bw of cyclopeptide E was administered orally to mice,and no obvious poisoning symptoms or signs of death were found during the 7-day observation period.Therefore,it is determined that the LD₅₀ of cyclopeptide E is greater than 5000 mg/kg·bw,which belongs to the actual non-toxic substance.At the same time,there were no significant differences in body weight,food intake,organ coefficient,blood routine,blood biochemistry,and pathological changes between the mice and the control group（p>0.05）.（3）In the genetic toxicity test,compared with the positive control group（cyclophosphamide）,the cyclopeptide E in each dose group had no significant effect on the micronucleus rate and sperm deformity rate of mice（p>0.05）,which showed that cyclopeptide E in flaxseed oil did not have mutagenic effects.（4）According to the conclusion of the acute toxicity test,the high,medium,and low doses in the subacute toxicity test were determined to be 3000 mg/kg·bw,2000 mg/kg·bw,and 1000 mg/kg·bw,respectively,for 28 consecutive days by gavage.The results showed that the mice in the high,medium,and low dose groups grew normally and showed no signs of death;There were no significant differences in weight growth trends,food utilization rates,and organ coefficients in each dose group of cyclopeptide E compared with the control group（p（29）0.05）;There was no significant difference in hematology and blood biochemical indexes compared with the control group（p（29）0.05）,and all the indexes were within the normal range;the observation results of pathological tissue sections in the high-dose group and the solvent control group showed that no obvious inflammation and pathological changes were found.In summary,the concentration of cyclopeptide E in flaxseed oil within 5000 mg/kg is non-toxic and can be utilized as a safe product development in the food field.