Immunomodulatory Activity Improvement of Pine Nut Peptides by a Pulsed Electric Field and Their Structure–Activity Relationships

Abstract

In this study, Alg-Gly-Ala-Val-Leu-His (RGAVLH) obtained from pine nut (Pinus koraiensis Sieb. et Zucc.) protein was chosen to investigate the phenomenon of immunomodulatory activity improvement upon pulsed electric field (PEF) processing. The influence of electric field intensity on immunomodulatory activity of RGAVLH was evaluated using RAW 264.7 cells. It was found that RGAVLH can not only significantly (p < 0.05) improve the capability of macrophage phagocytosis but also promote the production of nitric oxide. RGAVLH treated under an electric field intensity of 40 kV/cm exhibited the best immunomodulatory activity. The primary and secondary structures of PEF-treated peptides were analyzed by mid-infrared (MIR) spectroscopy, Raman spectroscopy, circular dichroism spectroscopy, and one-dimensional/two-dimensional nuclear magnetic resonance spectroscopy. After PEF treatment, the primary structure of RGAVLH was not influenced, as evaluated by MIR and Raman spectra. In addition, the content of β-sheet was decreased and active hydrogen was changed in PEF-treated RGAVLH solution. Moreover, the long-range connectivity between CαH (3.39 ppm) and NαH (8.24 ppm) was enhanced by PEF. Therefore, the improvement of the immunomodulatory activity of RGAVLH might result from the changes of the spatial state and spatial force in the peptide solution system.

Tryptophan targeted pulsed electric field treatment for enhanced immune activity in pine nut peptides

“…PEF processing could convert KWFCT structure β‐turn and random coil to each other, and KWFM structure β‐sheet, β‐turn, and random coil to each other. These were consistent with earlier reports that PEF could change the secondary structure of peptides and were shown in CD (Liang et al, 2017, 2018; Lin, Liang, Xing, et al, 2017; Lin, Liang, Xue, et al, 2017; Zhang et al, 2016; Zhang, Liang, et al, 2019). In the one‐dimensional NMR hydrogen spectrum, the active hydrogen on the peptide was shifted in many places, which was consistent with the previous research results.…”

“…Similar studies have been published. Zhang, Liang, et al (2019)) found that the long‐range correlation between C α H at 3.39 ppm and N α H at 8.24 ppm of the PEF‐treated RGAVLH was enhanced. The space force and state of the polypeptide solution could be mirrored by the short‐range and long‐range correlation between hydrogen.…”

“…Some of the following researches have used CD spectroscopy to show that PEF treatment could alter the secondary structure of peptides. Zhang, Liang, et al (2019)) demonstrated that PEF treatment could transform the random coils to β‐fold. Liang et al (2018) found that PEF treatment reduced the α‐helix contents of KCHQP and QCHKP, but increased those of KCHKP and QCHQP.…”

“…Liang et al (Liang et al, 2018) found that the chemical shifts at 14.46 ppm, 8.22 ppm, 7.87 ppm, 7.24 ppm, and 6.13 ppm attributable to hydrogen atoms of QCHKP shifted to the right by PEF. After PEF treatment, the active hydrogen at 7.356 ppm, 8.698 ppm, and 14.000 ppm in RGAVLH shifted (Zhang, Liang, et al, 2019). Zhang et al (2016) revealed that after the PEF treatment, the chemical environment of the hydrogen ion (proton) of peptide MMCTD was changed.…”

“…Phagocytosis assay was in view of the method of Zhang, Liang, and Zhao, (2019) with some improvements to discuss the effects of untreated and PEF‐treated pine nut peptides on phagocytic activity of macrophages. After cultivating, cells in 96‐well plate (10 5 cells/well) were incubated with pine nut peptide samples solution (100 μL/well) treated by PEF for 24 hr.…”