Torreya grandis Seed Polyphenols Protect RAW264.7 Macrophages by Inhibiting Oxidative Stress and Inflammation

Abstract

The seeds of Torreya grandis are rich in polyphenols, yet their chemical characteristics and biological activities require systematic elucidation. In this study, T. grandis seed polyphenols (TGSP) were prepared using ultrasound-assisted extraction (70% ethanol, solid-to-liquid ratio of 1:40 g/mL, 210 W power, 55°C, 50 min) coupled with AB-8 macroporous resin purification. The resulting TGSP were characterized by ultraviolet-visible (UV-Vis) spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Subsequently, their biological activities were systematically evaluated through in vitro chemical assays and in a cellular model. Structural analysis indicated that TGSP are abundant in gallic acid and catechins. TGSP exhibited selective scavenging activities against different free radicals, with half-maximal inhibitory concentrations (IC₅₀) for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS·+), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·), and hydroxyl radical (·OH) being 0.194 ± 0.015, 0.301 ± 0.020, and 1.013 ± 0.018 mg/mL, respectively. For comparison, the IC₅₀ values of vitamin C (VC) for ABTS·+ and DPPH· radicals were well below 0.1 mg/mL, and its IC₅₀ for ·OH radicals was 0.108 ± 0.011 mg/mL. At the cellular level, TGSP effectively inhibited the production of nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced RAW264.7 cells. Furthermore, TGSP significantly counteracted hydrogen peroxide (H₂O₂)-induced oxidative stress by reducing levels of reactive oxygen species (ROS) and malondialdehyde (MDA), restoring the activity of antioxidant enzymes such as superoxide dismutase (SOD), and suppressing caspase-3/9-mediated apoptosis. In conclusion, these findings demonstrate that T. grandis seed polyphenols exert significant cytoprotective effects through a multi-target mechanism, including direct free radical scavenging, inhibition of inflammation, and attenuation of oxidative stress-induced damage. This suggests their potential for development as functional food ingredients or natural pharmaceutical components.

Keywords: Torreya grandis seed; antioxidant activity; anti‐apoptosis; free radical scavenging; inflammation inhibition; polyphenols.

FIGURE 1

UV–Vis (A) and FT‐IR (B) spectra of TGSP.

FIGURE 2

Total ion chromatogram of TGSP in ESI negative ion mode LC–MS/MS.

FIGURE 3

Scavenging activity of TGSP against different types of free radicals. (A) ABTS + radical scavenging activity; (B) O₂ ⁻· superoxide anion radical scavenging activity; (C) OH hydroxyl radical scavenging activity; and (D) DPPH· radical scavenging activity.

FIGURE 4

Effects of TGSP on RAW264.7 cell viability. Values are presented as means ± SD of three independent experiments. Bars with different letters (a, b) indicate significant differences (p < 0.05).

FIGURE 5

Optimization of working concentrations for cell models. (A) Dose‐dependent stimulation of NO release by LPS. (B, C) Morphological transition of RAW264.7 cells from a resting (B) to an activated state (C) upon stimulation with 1 μg/mL LPS. (D) Dose‐dependent reduction in cell viability by H₂O₂. Values are presented as means ± SD of three independent experiments. Bars with different letters (a, b) indicate significant differences (p < 0.05).

FIGURE 6

TGSP inhibits LPS‐induced inflammatory cytokine production in RAW264.7 cells. (A) TNF‐α levels; (B) NO production; and (C) IL‐6 release. Values are presented as means ± SD of three independent experiments. Bars with different letters (a, b) indicate significant differences (p < 0.05).

FIGURE 7

Protective effects of TGSP against H₂O₂‐induced oxidative stress in RAW264.7 cells. (A) Cell viability; (B) SOD activity; (C) CAT activity; (D) GSH‐Px activity; (E) LDH release; (F) MDA content. Values are presented as means ± SD of three independent experiments. Bars with different letters (a, b) indicate significant differences (p < 0.05).

FIGURE 8

TGSP reduces H₂O₂‐induced ROS production and cell apoptosis in RAW264.7 cells. (A) DCFH‐DA fluorescence microscopy observation of ROS levels; (B) flow cytometry analysis of ROS production; (C) Annexin V‐FITC/PI double staining for cell apoptosis detection; (D) caspase‐3 activity; and (E) caspase‐9 activity. Values are presented as means ± SD of three independent experiments. Bars with different letters (a, b) indicate significant differences (p < 0.05).