Antioxidant and cholinesterase inhibitory activity of a new peptide from Ziziphus jujuba fruits

Abstract

Antioxidant agents and cholinesterase inhibitors are the foremost drugs for the treatment of Alzheimer's disease (AD). In this study, a new peptide from Ziziphus jujuba fruits was investigated for its inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes as well as antioxidant activity. This peptide was introduced as a new peptide and named Snakin-Z. The Snakin-Z displayed considerable cholinesterase inhibition against AChE and BChE. The half maximal inhibitory concentration (IC50) values of Snakin-Z against AChE and BChE are 0.58 ± 0.08 and 0.72 ± 0.085 mg/mL, respectively. This peptide has 80% enzyme inhibitory activity on AChE and BChE at 1.5 mg/mL. The Snakin-Z also had the high antioxidant activity (IC50 = 0.75 ± 0.09 mg/mL). Thus, it is suggested that Snakin-Z may be beneficial in the treatment of AD. However, more detailed researches are still required as in vivo testing its anticholinesterase and antioxidant activities.

Keywords: Alzheimer’s disease; Ziziphus jujuba fruits; acetylcholinesterase; antioxidant; butyrylcholinesterase.

Figure 1.

Purification of peptides from Zizyphus jujuba fruit. A, Sephadex G-50 gel filtration of plant extract was applied on a Sephadex G-50 column equilibrated with phosphate buffer (0.1 mol/L, pH 6.0). B, The peak 4 (indicated with asterisk), with anticholinesterase activity from Sephadex G-50, was further purified on a C18 reverse phase-high-performance liquid chromatography (RP-HPLC) column. A 400-μL aliquot of filtrated extract from gel filtration was loaded onto a semipreparative C18 reverse phase column. Elution was performed using solution A (0.1% TFA in water), combined with a 5% to 65% gradient of solution B (0.098% TFA in acetonitrile) over a period of 70 minutes, at a flow rate of 1 mL/min. The absorbance was monitored at 220 nm, and the fractions were tested for anticholinesterase activity. The active peak has been indicated by an arrow. This peak was named Z (Zizyphus jujuba). TFA indicates trifluoroacetic acid.

Figure 2.

DPPH radical scavenging activity (%) of Snakin-Z from Ziziphus jujuba fruits. butylated hydroxyanisole (BHA) is positive control.

Figure 3.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities (%) of Snakin-Z from Ziziphus jujuba fruits.

Figure 4.

Tricine-SDS-PAGE of raw extract (1), active fraction (peak 4) from gel filtration column (2), purified peptide from C18 reverse phase-high-performance liquid chromatography (RP-HPLC) column (3), and size marker (4). A, Trypsinogen (23 kDa); B, lysozyme (14 kDa); and C, insulin (5.8 kDa).

Figure 5.

Identification of the molecular mass and amino acid sequence of the new peptide from the fruit of Ziziphus jujube using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) spectrometer.

Figure 6.

The alignment and phylogenetic tree of Snakin-Z. A, The alignment of amino acid sequences of Snakin-Z with the sequences of other antimicrobial peptides. The alignment was carried out with CLC Main Work Bench Ver.5.5 software. B, Phylogenetic tree of Snakin-Z. Amino acid sequences of the 10 reference peptides obtained from the APD database were incorporated into the tree using the neighbor-joining method. The name of each sequence is typed at the end of the corresponding branch. Reliability of the tree was assessed by bootstrap analysis with 100 replications. The substitutions per amino acid position are typed above each branch.