Xingrong Ju

Antihypertensive and free radical scavenging properties of enzymatic rapeseed protein hydrolysates

In this study, rapeseed protein isolate (RPI) was digested with various proteases to produce rapeseed protein hydrolysates (RPHs), which were then separated into different peptide fractions (<1, 1-3, 3-5, and 5-10kDa) by membrane ultrafiltration. Membrane fractionation showed that peptides with sizes <3 kDa had significantly (p<0.05) reduced surface hydrophobicity when compared to the RPHs and peptide fractions with sizes >3 kDa. In contrast, the <3 kDa peptides showed significantly (p<0.05) higher oxygen radical scavenging ability when compared to the >3 kDa peptides and RPHs. In vitro inhibition of angiotensin I-converting enzyme (ACE) was significantly (p<0.05) higher for the Thermolysin, Proteinase K and Alcalase RPHs when compared to the pepsin+pancreatin (PP) and Flavourzyme RPHs. The Alcalase RPH had significantly (p<0.05) higher renin inhibition among the RPHs, while with the exception of Thermolysin, the 5-10 kDa peptide fraction had the least renin-inhibitory ability when compared to the <5 kDa peptide fractions. Oral administration (100mg/kg body weight) of the RPHs and RPI to spontaneously hypertensive rats (SHR) showed the Alcalase RPH to be the most effective in blood pressure (BP) reduction (∼24 mm Hg) while Proteinase K RPH was the least effective (∼5 mm Hg) after 8h. However, the PP RPH had the most prolonged effect with BP reduction of ∼20 mm Hg after 24h of oral administration. We conclude that the strong BP-lowering ability of Alcalase and PP RPHs could be due to high resistance of the peptides to structural degradation coupled with high absorption rate within the gastrointestinal tract.

Glycinyl-histidinyl-serine (GHS), a novel rapeseed protein-derived peptide has blood pressure-lowering effect in spontaneously hypertensive rats.

A novel antihypertensive peptide (Gly-His-Ser or GHS) with dual inhibition of angiotensin I-converting enzyme (ACE) and renin activities was isolated from the 3 kDa membrane ultrafiltration permeate of a pepsin+pancreatin rapeseed protein digest. The IC50 values of GHS were 0.52 ± 0.01 mg/mL and 0.32 ± 0.01 mg/mL for ACE and renin inhibitions, respectively, which are 1.5 times the ACE inhibition and 3.5 times the renin inhibition of the 3 kDa permeate. Oral administration (30 mg/kg body weight) to spontaneously hypertensive rats showed GHS to be an effective hypotensive agent with maximum blood pressure reduction of -17.29 ± 2.47 mmHg after 6 h. In contrast, the 3 kDa permeate exhibited a maximum of -21.29 ± 9.29 mmHg after 4 h, although at a relatively higher dose of 100 mg/kg body weight). GHS inhibited ACE and renin activities noncompetitively, but the renin inhibition became uncompetitive at a higher peptide concentration.

Evaluating Molecular Mechanism of Hypotensive Peptides Interactions with Renin and Angiotensin Converting Enzyme

Our previous study showed that three rapeseed protein-derived peptides (TF, LY and RALP) inhibited the in vitro activities of angiotensin converting enzyme (ACE) and renin. Oral administration of these peptides to spontaneously hypertensive rats led to reductions in systolic blood pressure. In the present work, we examined the potential molecular mechanisms responsible for the ACE- and renin-inhibitory activities of these peptides. Enzyme inhibition kinetics showed competitive, non-competitive and mixed-type peptide-dependent inhibition of renin and ACE activities. Intrinsic fluorescence intensity data showed that LY and RALP have stronger binding effects on ACE molecule compared to that of TF. LY and RALP showed the highest inhibition of ACE and renin activities, respectively. Circular dichroism data showed that the inhibitory mechanism involved extensive peptide-dependent reductions in α-helix and β-sheet fractions of ACE and renin protein conformations. Molecular docking studies confirmed that the higher renin-inhibitory activity of RALP may be due to formation of several hydrogen bonds (H-bonds) with the enzyme’s active site residues. The rapeseed peptides inhibited renin and ACE activities mostly through binding to enzyme active site or non-active sites and forming extensive H-bonds that distorted the normal configuration required for catalysis. Data presented from this work could enhance development of highly potent antihypertensive natural peptides or peptidomimetics.

Structural characterization of phenolic compounds and antioxidant activity of the phenolic-rich fraction from defatted adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) seed meal

The current study aims to investigate the antioxidant activities of various extracts from defatted adlay seed meal (DASM) based on the oxygen radical absorbance capacity (ORAC) assay, peroxyl radical scavenging capacity (PSC) assay and cellular antioxidant activity (CAA) assay. Of all the fractions, the n-butanol fraction exhibited the highest antioxidant activity, followed by crude acetone extract and aqueous fractions. Of the three sub-fractions obtained by Sephadex LH-20 chromatography, sub-fraction 3 possessed the highest antioxidant activity and total phenolic content. There was a strong positive correlation between the total phenolic content and the antioxidant activity. Based on HPLC-DAD-ESI-MS/MS analysis, the most abundant phenolic acid in sub-fraction 3 of DASM was ferulic acid at 67.28 mg/g, whereas the predominant flavonoid was rutin at 41.11 mg/g. Of the major individual compounds in sub-fraction 3, p-coumaric acid exhibited the highest ORAC values, and quercetin exhibited the highest PSC values and CAA values.

Selective separation and concentration of antihypertensive peptides from rapeseed protein hydrolysate by electrodialysis with ultrafiltration membranes.

Rapeseed protein isolate was subjected to alcalase digestion to obtain a protein hydrolysate that was separated into peptide fractions using electrodialysis with ultrafiltration membrane (EDUF) technology. The EDUF process (6h duration) led to isolation of three peptide fractions: anionic (recovered in KCl-1 compartment), cationic (recovered in KCl-2 compartment), and those that remained in the feed compartment, which was labeled final rapeseed protein hydrolysate (FRPH). As expected the KCl-1 peptides were enriched in negatively-charged (43.57%) while KCl-2 contained high contents of positively-charged (28.35%) amino acids. All the samples inhibited angiotensin converting enzyme (ACE) and renin activities in dose-dependent manner with original rapeseed protein hydrolysate having the least ACE-inhibitory IC50 value of 0.0932±0.0037 mg/mL while FRPH and KCl-2 had least renin-inhibitory IC50 values of 0.47±0.05 and 0.55±0.06 mg/mL, respectively. Six hours after oral administration (100 mg/kg body weight) to spontaneously hypertensive rats, the FRPH produced the maximum systolic blood pressure reduction of -51 mmHg.

Transepithelial Transport of YWDHNNPQIR and Its Metabolic Fate with Cytoprotection against Oxidative Stress in Human Intestinal Caco-2 Cells

Studies on antioxidant peptides extracted from foodstuff sources have included not only experiments to elucidate their chemical characteristics but also to investigate their bioavailability and intracellular mechanisms. This study was designed to clarify the absorption and antioxidative activity of YWDHNNPQIR (named RAP), which is derived from rapeseed protein using a Caco-2 cell transwell model. Results showed that 0.8% RAP (C0 = 0.2 mM, t = 90 min) could maintain the original structure across the Caco-2 cell monolayers via the intracellular transcytosis pathway, and the apparent drug absorption rate (Papp) was (6.6 ± 1.24) × 10-7 cm/s. Three main fragments (WDHNNPQIR, DHNNPQIR, and YWDHNNPQ) and five modified peptides derived from RAP were found in both the apical and basolateral side of the Caco-2 cell transwell model. Among these new metabolites, WDHNNPQIR had the greatest antioxidative activity in Caco-2 cells apart from the DPPH assay. With a RAP concentration of 200 μM, there were significant differences in four antioxidative indicators (T-AOC, GSH-Px, SOD, and MDA) compared to the oxidative stress control (P < 0.05). In addition, RAP may also influence apoptosis of the Caco-2 cells, which was caused by AAPH-induced oxidative damage.

Study on Antioxidant Activity and Amino Acid Analysis of Rapeseed Protein Hydrolysates

Alkaline extraction followed by acid precipitation were employed to extract rapeseed protein and, Alcalase 2.4 L was used to obtain rapeseed protein hydrolysates. Three groups of rapeseed protein hydrolysates were obtained by purifying with membrane ultrafiltration and a Sephacryl S-100HR gel column. The antioxidant activities were then determined. Group 3 had the best antioxidant activities according to the oxygen radical absorbance capacity, peroxyl radical-scavenging capacity, and cellular antioxidant activity assays, with the following antioxidant values: an oxygen radical absorbance capacity value of 1610 ± 113 µmol TE/(g sample), a peroxyl radical-scavenging capacity value of 622 ± 30 mg VC/(100 g sample), and a cellular antioxidant activity value of 25 ± 2 µmol QE/(g sample) and corresponding EC50 value of 58 ± 3 µg/mL. Six peaks of group 3 were collected and well separated by reversed phase–high-performance liquid chromatography. Peak 5 were identified to exhibit a higher antioxidant activity, the amino acid sequence of which was found to be Trp-Ile (Leu)-Tyr, as determined by liquid chromatography–mass spectrometry.

A safe, efficient and simple technique for the removal of cadmium from brown rice flour with citric acid and analyzed by inductively coupled plasma mass spectrometry

Rice (Oryza sativa L.), an important cereal crop, has been seriously contaminated by cadmium (Cd) in the world. In this study, we compared the effects of three organic acids (citric acid, malic acid and tartaric acid) which are all approved food additives on the removal of Cd and developed a safe, efficient and simple technique to remove Cd from contaminated brown rice flour using citric acid (CA). The optimal conditions for citric acid removal of Cd were determined by a three-level Box–Behnken design with three factors (CA concentration, treatment temperature and treatment time) under the condition that the liquid–solid ratio was 12 mL g−1 and the shaking speed was 150 rpm, and the results were as follows: CA concentration, 0.08 mol L−1; treatment temperature, 45 °C; and treatment time, 53.09 min. Under these conditions, the removal efficiency of Cd was 94.28 ± 0.06%, which agreed well with the predicted value of 94.75%. Moreover, the contents of the main chemical components such as protein, starch and fat in the brown rice flour were still very high after citric acid treatment, indicating that this technique did not affect the quality of brown rice flour. This technique was also used in other brown rice flour samples and satisfactory results were obtained. Therefore, this technique is efficient in removing Cd and may have the potential to be used in the food industry to solve the Cd-rich rice problem in the world.

Polyelectrolyte complex nanoparticles from chitosan and acylated rapeseed cruciferin protein for curcumin delivery

Curcumin is a polyphenol that exhibits several biological activities, but its low aqueous solubility results in low bioavailability. To improve curcumin bioavailability, this study has focused on developing a polyelectrolyte complexation method to form layer-by-layer assembled nanoparticles, for curcumin delivery, with positively charged chitosan (CS) and negatively charged acylated cruciferin (ACRU), a rapeseed globulin. Nanoparticles (NPs) were prepared from ACRU and CS (2:1) at pH 5.7. Three samples with weight of 5%, 10%, and 15% of curcumin, respectively, in ACRU/CS carrier were prepared. To verify the stability of the NPs, encapsulation efficiency and size of the 5% Cur-ACRU/CS NPs were determined at intervals of 5 days in a one month period. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction, and differential scanning calorimetry confirmed the electrostatic interaction and hydrogen bond formation between the carrier and core. The result showed that hollow ACRU/CS nanocapsules (ACRU/CS NPs) and curcumin-loaded ACRU/CS nanoparticles (Cur-ACRU/CS NPs) were homogenized spherical with average sizes of 200-450 nm and zeta potential of +15 mV. Encapsulation and loading efficiencies were 72% and 5.4%, respectively. In vitro release study using simulated gastro (SGF) and intestinal fluids (SIF) showed controlled release of curcumin in 6 h of exposure. Additionally, the Cur-ACRU/CS NPs are nontoxic to cultured Caco-2 cells, and the permeability assay indicated that Cur-ACRU/CS NPs had improved permeability efficiency of free curcumin through the Caco-2 cell monolayer. The findings suggest that ACRU/CS NPs can be used for encapsulation and delivery of curcumin in functional foods.

The preparation and physiochemical characterization of rapeseed protein hydrolysate-chitosan composite films

The composite films were prepared by mixing rapeseed protein hydrolysate with chitosan. Upon increasing the degree of hydrolysis, rapeseed protein enhanced its compatibility with chitosan, thus making composite films denser. The tensile strength of films was increased from 16.04 to 23.46 MPa with increasing the degree of hydrolysis from 0% to 12%. Moreover, addition of chitosan enhances the mechanical properties of the rapeseed protein films, the α-helix content in the secondary structure of the rapeseed protein from 15.4% to 25.0%. And it is hydrogen bonding, the main force between two components that contributed to good compatibility, which supported by analyses of Fourier transform infrared spectroscopy and scanning electron microscopy. The results of the antibacterial properties of the composite film with 12% degree of hydrolysis were better compared with the chitosan film. Taken together, our results provide insights for the further application of rapeseed protein in making edible films.