Purification, Characterization and Mechanistic Evaluation of Angiotensin Converting Enzyme Inhibitory Peptides Derived from Zizyphus Jujuba Fruit

Abstract

The synthetic Angiotensin Converting Enzyme (ACE) inhibitors have side effects and hence demands for natural ACE inhibitors have been rising. The aim of this study is to purify and introduce natural ACE inhibitors extracted from Zizyphus jujuba fruits. Proteins from Zizyphus jujuba were lysed by trypsin, papain and their combination. Acquired peptides were purified and evaluated for ACE inhibitory activity. Peptide fractions with inhibitory activity were sequenced using tandem mass spectrometry. To elucidate the mode of peptide binding to ACE, homology modeling, molecular docking and molecular dynamics simulation were performed. Amino acid sequence of F2 and F4 peptides, which were the most active hydrolysates, were determined to be IER and IGK with the IC 50 values of 0.060 and 0.072 mg/ ml, respectively. Results obtained by computational analysis revealed that similar to the common ACE competitive inhibitors such as captopril, IER tripeptide binds to the enzyme active site, in vicinity of the zinc binding site, and occupies the S1 and S2' subsites. Binding occurs through hydrogen bonding with Gln293, Lys522, His524, Tyr531 and also several hydrophobic interactions. Collectively, these findings indicate that IER tripeptide inhibits the rabbit ACE enzyme through a competitive mechanism of inhibition with IC 50 values in the millimolar range.Angiotensin converting enzyme (ACE) inhibitors are a new class of drugs which are critical in the treatment of hypertension and heart failure 1 . Although the synthetic drugs are often prescribed due to their well-known role and convenience of access, they have undesirable effects, such as extreme low blood pressure, cough, bad tastes and allergic reactions. Contrariwise, natural inhibitors regulate hypertension under mild conditions without significant side effects; hence, they can be considered as worthy alternatives to synthetic drugs 2 .ACE inhibitory peptides are the best known amongst various classes of regulatory bioactive peptides due to the development of functional foods with homeostasis role in the human body 3 . ACE is a Zn-metallopeptidase that removes a dipeptide from the decapeptide angiotensin-I to produce the potent vasoconstriction octapeptide angiotensin-II 4 . Several peptides originated from animal or plant proteins exhibit significant therapeutic/ regulatory performances like antihypertensive, antimicrobial, antioxidant, immunoregulatory, anticancer activities, etc 3 .Investigations to find more bioactive peptides, especially multifunctional ones have attracted the attention of researchers. The bioactive peptides are often inactive in their natural forms and when released by enzymatic hydrolysis, display their biological activity 3,5 . So far, several ACE inhibitory peptides have been reported from hydrolysis of different food proteins, such as hen egg white lysozyme 4 , fish 6 , corn 7 , soybean 8 , milk 9 , casein 10 , etc. These peptides are good candidates to regulate blood pressure and also to balance fluid and electrolytes in mammals. Scientific Rep...

Physicochemical and antioxidant activity of fruit harvested from eight jujube (Ziziphus jujuba Mill.) cultivars at different development stages

“…Jujube fruit has been reported to be high in carbohydrates, organic acids, amino acids 4 , vitamin C and minerals 5 . Recent studies have reported that jujube fruit provides health benefits such as antioxidant and anticancer, hepatoprotective, gastrointestinal protective, anti-inflammatory, anti-insomnia, immunostimulating, and neuroprotective effects 6 . These medicinal properties have been shown to be closely related to the composition of jujube’s bioactive components.…”

Characterization of ACE inhibitory and antioxidant peptides in yak and cow milk hard chhurpi cheese of the Sikkim Himalayan region

“…Captopril is a chemically synthesized l -proline derivative and is widely used in the therapy of hypertension ( Memarpoor-Yazdi, Zare-Zardini, Mogharrab, & Navapour, 2020 ). About 73.65 % ACE-inhibition was observed for the positive control captopril at the concentration of 10 ng/ml.…”

“…The catalytic site of ACE includes the zinc binding domain (His387, His383, and Glu411), and the three active site pockets, S1 (Ala354, Glu384, and Tyr523), S1′ (Glu162), and S2 (Gln281, His353, Lys511, His513, Tyr 520, and His523) that are not only critical for ACE activity but also the main binding targets for ACE inhibitory drugs ( Xue et al, 2021 ). Captopril, the positive control used as ACE-inhibitor in this study, shows non-bond interaction with the ACE S2 residues, Gln281, His353, His513, Lys511, Tyr520, and Glu 384, of the S1 active site pocket ( Memarpoor-Yazdi et al, 2020 ). Non-bond interactions with identical residues were observed for previously reported and predicted ACE-inhibitory peptides identified in the present study.…”

Detection of Decarboxylated Amino Acids after <i>in Vitro</i> Protease Digestion of the Hydrophilic Fraction of Crude Drug Extracts

“…inermis. 29) They used trypsin and papain (plant protease) for in vitro digestion and found that a protease-resistant tripeptide (Ile-Glu-Arg) competitively inhibited rabbit angiotensin converting enzyme. Therefore, the current study may be applied to pharmacological studies on proteins and peptides and facilitate understanding how crude drugs and Kampo medicine manifest their effects in vivo.…”