Screening And Mechanism Of Action Of ACE Inhibitory Peptides From The Symbiont Of Boletus Chrysosporium Parasitoid

Hypertension is a common cardiovascular and cerebrovascular disease,but drug treatment is accompanied by many adverse reactions.Therefore,it is of great significance to find a natural substance with antihypertensive activity.In recent years,food-derived angiotensin-converting enzyme（ACE）inhibitory peptides have become a highly promising peptide due to their strong blood pressure lowering activity,good absorption,and high safety.In this study,the composition of symbiot Boletus griseus-Hypomyces chrysospermus（BGHC）was analyzed.The key bioactive peptides were screened from the methanol extract of the BGHC.The absorption,distribution,metabolism,excretion and toxicity（ADMET）properties,activity,mechanism of action,simulated gastrointestinal digestion stability and potential antihypertensive targets of the key peptides were studied.The main research contents and results are as follows:（1）The composition of the BGHC was analyzed.The results showed that the contents of crude protein,water-soluble polysaccharides,ash,total free amino acids and polyphenols in the BGHC were 16.51%,23.84%,5.61%,3.10%and 11.73 mg gallic acid/g Dry weight（DW）,respectively.Among the 20 free amino acids detected,the total content of umami amino acids and sweet amino acids was 4.15 mg/g DW,accounting for 31.92%of the total free amino acids.The content of essential amino acids in hydrolyzed amino acids was higher,accounting for 38%of the total essential amino acids.The methanol extract of the BGHC had high ACE inhibitory activity,and its IC₅₀value was 20.09μg/m L.Ultra Performance Liquid Chromatography-Quadrupole-Orbitrap-massspectrometry/mass spectrometry（UPLC-Q-Orbitrap-MS²）was used to identify 22 peptides with average local confidence scores≥85%from the methanol extract.（2）Based on the identified 22 peptides,two peptides KYPHVF and LFRPE with potential ACE inhibitory（ACEI）activity were screened by Peptide Ranker online tool and BIOPEP-UWM database.The prediction results of ADMET properties show that both KYPHVF and LFRPE have good ADMET properties.The ACE inhibition mechanism of KYPHVF and LFRPE was explored by molecular docking.The results showed that both peptides could change the conformation of ACE and exert ACEI activity by forming hydrogen bonds and hydrophobic interactions with ACE active sites.Finally,in vitro ACEI activity experiments confirmed that KYPHVF and LFRPE were ACEI peptides with IC₅₀values of 5.99 and 58.15μM,respectively.（3）In vitro simulated gastrointestinal digestion experiments showed that KYPHVF maintained structural integrity,while LFRPE was degraded into FRPE.The transport rate and peptide sequence of KYPHVF and LFRPE through Caco-2 cell monolayer were analyzed by UPLC-Q-Orbitrap-MS².The results showed that the transport rates of KYPHVF and LFRPE were 2.00%and 2.24%,respectively.After transport through Caco-2 cell monolayer,KYPHVF and LFRPE were partially degraded.An in vivo blood pressure lowering experiment in spontaneously hypertensive rats（SHRs）with a single gavage,demonstrating that both KYPHVF and LFRPE can effectively reduce the blood pressure of SHRs in vivo.Both of them,KYPHVF exerts its antihypertensive effect by reducing the ACE content in serum and kidney of SHRs.（4）The Pharm Mapper database was used to predict the targets of KYPHVF and LFRPE,and the hypertension-related target set was obtained through the Gene Cards and CTD databases.The targets of KYPHVF and LFRPE were mapped to the hypertension-related target sets,and 44 potential antihypertensive targets of KYPHVF and 46 potential antihypertensive targets of LFRPE were obtained.The protein interaction network of potential antihypertensive targets of KYPHVF and LFRPE was analyzed by STRING database and Cytoscape software.It was found that many targets such as hydrolase,transferase,oxidoreductase,signaling protein,and growth factor may be involved in the antihypertensive process of KYPHVF and LFRPE.The results of GO functional enrichment analysis showed that KYPHVF and LFRPE could play various molecular functions in different cellular components by regulating various biological processes to achieve antihypertensive effect.Cytoscape software was used to construct a target-pathway network,and 12 potential key antihypertensive targets of KYPHVF and 12 potential key antihypertensive targets of LFRPE were screened out.The results showed that KYPHVF may play a role in lowering blood pressure mainly by targeting MAPK14,MAP2K1,EGFR,SRC,RHOA,JAK2,CASP3,MMP9,STAT1,NOS3 and IGF1.LFRPE may play a role in lowering blood pressure mainly by targeting MAPK14,EGFR,SRC,TGFB2,RHOA,JAK2,IGF1,STAT1,MMP9 and KDR.