Preparation And Mechanism Of Anti-Anxious Depression Peptide

Anxiety and depression are complex psychiatric disorders that often occur together with a high comorbidity.5-HT is closely associated with the etiology of depression and/or anxiety.Most of the drugs currently used in clinical practice alleviate and treat anxiety and depression-like symptoms by increasing the level of5-HT in the synaptic cleft,but these drugs have obvious side effects and even toxic effects.Food-derived bioactive peptides have become research hotspots for the development of specific functional foods in recent years due to their diverse biological activities,extensive sources,simple preparation process,and non-toxic side effects.Trp is a precursor of 5-HT,the availability of Trp in the brain affects 5-TH levels in the synaptic gap.The peripheral 5-HT cannot go across the blood–brain barrier,as a result,the concentration of 5-HT in the brain is decreased?which is a contributor to the etiology of anxiety/depression?.Trp influx to the brain and its availability in the brain are affected by a number of factors.The competition of Trp with the five large neutral amino acids?5LNAAs?,Val,Ile,Leu,Phe and Tyr during its passage through the blood-brain barrier by carriers.In mammals,only a small portion of Trp is utilized for the 5-HT synthesis while the majority of Trp is catabolized via the kynurenic pathway to bioactive substances to combat stress and inflammation.Therefore,increasing the ratio of plasma Trp to LNAAs?ie,the Trp index?and promoting the conversion of brain Trp to the 5-HT metabolic pathway is the key to increasing the bioavailability of Trp.Accordingly,whey protein was used as the raw material for enzymatic hydrolysis to prepare hydrolysate with a high Trp/5LNAAs ratio?WPH?.The enzymatic synthesis of?-[Glu]_n-Trp?EW?was conducted through mixing Gln and Trp in capped containers,with the catalyst L-glutaminase from Bacillus amyloliquefacien.The objective of this study was to investigate the effect of WPH and EW on 5-HT secretion in Zebrafish and on chronic restraint stress?CRS?-corticosterone?CORT?-induced anxiety/depression-like state in C57BL/6 mice.The aim was to provide theoretical basis and method guidance for the industrial production of WPH and EW dietary supplements and the mechanism of improving anxious depression.The main research contents and results of this paper are as follows:?1?WPH preparation and tryptophan peptide structure identification:Whey protein?protein content:80.37%?Trp peptides was conducted by trypsin under conditions as follows:whey protein to water as 1:9?m/v?,p H 8.0,15.21 U/g enzyme added,hydrolysis at 50?for 24 h.The amino acid analyses of trypsin had the highest Trp/5LNAAs ratio?17.38%?.Five Trp oligopeptide?Val-Ala-Gly-Thr-Trp?VAGTW?,Val-Ala-Gly-Thr-Trp-Tyr?VAGTWY?,Gly-Thr-Trp?GTW??were identified in whey protein hydrolysate.Preparation of Trp peptides and its structure identification:using Trp as?-glutamyl acceptor and Gln as donor,?-[glu]_n-Trp was synthesized by glutaminase derived from Bacillus amyloliquefaciens.The results showed that the total synthesis rate of?-[glu]_n-Trp is up to 79.05%and was obtained at p H 10.0,37.0?,enzyme addition 0.1%?m/v?,substrate concentration 0.1 mol/L,and the addition ratio of Trp and Gln is 1:2.Identification of the tryptophan-glutamine reaction system by UPLC-ESI-MS/MS and manual sequencing de novo software including?-glu-Trp??-EW???-[glu]₂-Trp??-EEW???-[glu]₃-Trp??-EEW???-[glu]₄-Trp??-EEEEW?.The total conversion of EW reached 79.05%?w/w?.?2?The antioxidant activity of Trp peptide and its effect on 5-HT secretion in zebrafish were studied.The results show that both WPH and EW have good antioxidant activity,WPH and VAGTW,VAGTWY,GTW,SAWY and WYS have strong free radical scavenging ability,EW and?-EW,?-EEW,?-EEEW and?-EEEEW has good metal ion chelating ability and total antioxidant activity.Zebrafish model biological experiments show that WPH,EW,and Trp peptide monomer standards can effectively improve the efficacy of normal zebrafish body weight gain index,Trp concentration,TPH enzyme activity and 5-HT level,with EW has the better effect.In addition,most Trp peptide monomer standards are superior to EW and WPH.The effect of?-EEEW was the best in Trp peptide monomer standard of EW source,and the effect of VAGTW was the best in Trp peptide monomer standard of WPH source.?3?The effects of WPH and EW on chronic restraint stress?CRS?-corticosterone?CORT?-induced anxious depression-like state in C57BL/6 mice were studied.The results showed that WPH?dose 0.5,1.0 or 2.0 mg/g·d?and EW?dose 2.0,5.0 or 10.0mg/kg·d?reversed slow weight gain,behavioral dysfunctions and low 5-HT levels in the central nervous system,with EWH acting more effectively.The levels of 5-HT in the prefrontal cortex,hippocampus and hypothalamus of the mice in the EW were significantly higher than those in the WPH.?4?Trp peptide down-regulates KYN metabolic pathway through intervention on inflammatory factors was investigated.The KYN metabolic pathway down-regulation may mediate by inhibiting IDO activity via WPH and EW anti-inflammatory effect.Furthermore,Trp depletion and accumulation of neuroactive metabolites can be avoided,and the bioavailability of Trp can be improved.?5?The mechanism of Trp peptide improvement in anxious depression was studied and analyzed.EW treatment increased brain-derived 5-HT levels and reversed the detrimental effects on the hippocampus and hypothalamus,with the high dose being the most effective.WPH and EW may reduce malondialdehyde-induced damage to 5-HT neurons and central neurons by scavenging free radicals.WPH and EW can up-regulate m RNA expression of BDNF/Trk B signaling pathway,promote neuron regeneration and repair.The neuroprotective effect further promotes the synthesis of 5-HT in the central nervous system and increases the level of 5-HT in the synaptic cleft.