Peptides Identified And Inhibitory Activities Analysis On DPP-? And ACE From Tea(camellia Sinensis)fresh Leaves And Black Tea

Bioactive peptides in plants are endogenous substances which play an important role in regulating the development of resistance to pathogens and have excellent potential for applications in health management.Many food-derived bio active peptides with antidiabetic,antihypertensive,antioxidant,and anticancer effects have been identified.The Tea plant(Camellia sinensis)is one of the most economically important crops in south China.Tea is the most popular beverage(second only to water)and is rich in functional compounds.However,its functional bioactive peptides are seldom studied.In this study comparative peptidomics was used,for the first time,to investigate endogenous peptides derived from the tea cultivars HJY(Huang Jin Ya)and LJ-43(Long Jing-43).Peptides were identified and classified by characteristic and function.Dipeptidyl peptidase ?(DPP-?)and angiotensin 1-converting enzyme(ACE)are key enzymes in the pathogenesis of diabetes and hypertension,respectively.Molecular simulation,virtual screening,peptidomics,and functional verification were used to identify DPP-? and ACE inhibitory peptides in black tea and tea plant.Peptidomics identified 708 peptides derived from HJY and LJ-43.COG and pathway analyses revealed that most of their precursor proteins related to carbohydrate transport and metabolism.Differential analysis of the two cultivars showed there were thirty-eight peptides with differential expression.Twenty-five were down-regulated and thirteen up-regulated in HJY compared with LJ-43.The functions of the precursor proteins explain this difference in composition.Several polypeptides with the greatest differential expression were related to ribulose-1,5-bisphosphate carboxylase-oxygenase,raffinose synthetase,and ribosomal protein.The HJY and LJ-43 peptides were searched for ACE inhibitory peptides.Eighty-seven peptides with potential inhibitory effects were selected by virtual screening.Ten were synthesized to verify their inhibitory effect in vitro.All ten showed inhibitory potency with three(P1-P3)having significantly lower IC50 values.Lineweaver-Burk plots showed these three inhibited ACE in a non-competitive manner.Molecular docking and molecular dynamics simulation showed that all three bound to residues in the active pockets of ACE.P3 showed the best binding stability and bio availability prediction showed it was the most specific peptideFood components,including bioactive peptides,have recently been proposed as DPP-? inhibitors with minimal side effects.Four peptides identified in black tea showed DPP-? inhibitory activity in vitro,of which peptide ?(976 Da;sequence:AGFAGDDAPR)had the greatest impact.After administration of peptide ?(400 mg/day for 57 d)to streptozotocin-induced hyperglycemic mice,the concentration of glucagon-like peptide-1 in the blood increased from 9.85±1.96 pM to 19.22±6.79 pM and the insulin level increased 4.3-fold.Immunohistochemistry revealed the improved function of pancreatic beta-cells and suppressed proliferation of pancreatic alpha-cells.This study provides new insight into black tea as a potential source of DPP-? inhibitory peptides for the management of type 2 diabetesThe inhibitory mechanisms of tea peptides were elucidated by analysis of the affinities between the peptides and ACE as well as stabilities of the complexes using in vitro and in silico methods.Four peptides(?,?,?,and ?)were examined for ACE inhibition and kinetics.IC50 values were 210.03±18.29,178.91±5.18,196.31± 2.87 and 121.11 ± 3.38 ?M,respectively.Lineweaver-Burk plots showed peptides ?,?,and ? inhibited ACE in an uncompetitive manner which requires the presence of substrate(peptide ? acted non-competitively,for which substrate is not necessary)Docking simulations showed that the peptides did not bind to the active sites of ACE,indicating they were allosteric inhibitors.Binding free energies calculated from molecular dynamic simulation were-22.65,-78.36,-52.10,and-86.66 kcal/mol,respectively.The low IC50 and binding free energy of peptide ? may be attributed to strong hydrogen bonding and changes in the flexibility and unfolding of ACE.These four bioactive peptides with ACE inhibitory ability may be utilized as novel functional food ingredients.