Hemicellulosic Polysaccharides From Bamboo Leaves Promoted by Phosphotungstic Acids and Its Attenuation of Oxidative Stress in HepG2 Cells

Abstract

In this work, we exploited an efficient method to release hemicellulosic polysaccharides (BLHP) from bamboo (Phyllostachys pubescens Mazel) leaves assisted by a small amount of phosphotungstic acid. Structural unit analysis proved that BLHP-A1 and BLHP-B1 samples possessed abundant low-branch chains in →4)-β-_D-Xylp-(1→ skeleton mainly consisting of Xylp, Manp, Glcp, Galp, and Araf residues. According to the results of the antioxidant activity assays in vitro, both of the two fractions demonstrated the activity for scavenging DPPH⋅ and ABTS⁺ radicals and exhibited relatively a high reducing ability compared to the recently reported polysaccharides. Moreover, the antioxidant activities of purified polysaccharides were evaluated against H₂O₂-induced oxidative stress damage in HepG2 cells. BLHP-B1 showed more activity for preventing damages from H₂O₂ in HepG2 cells by improving the enzyme activities of SOD, CAT, and GSH-Px and decreasing the production of MDA as well as suppressing reactive oxygen species (ROS) formation. This study implied that BLHP could demonstrate its attenuation ability for oxidative stress in HepG2 cells.

Keywords: HepG2 cells; bamboo leaves; hetero-polysaccharides; oxidative stress; phosphotungstic acid.

FIGURE 1

Purification of the BLHP samples: (A) the crude polysaccharides were applied to DEAE-Sepharose fast flow column (2.0 cm × 40 cm, Cl^– form) to produce BLHP-A and BLHP-B fractions; (B) DEAE Sephacryl S-300 column (2.0 cm × 40 cm) was applied to purify BLHP-A sample; (C) DEAE Sephacryl S-300 column (2.0 cm × 40 cm) was applied to purify BLHP-B sample; (D) chromatogram of the molar mass distribution of BLHP-A1 and BLHP-B1 detected by GPC.

FIGURE 2

The monosaccharide content in crude polysaccharides, BLHP-A1 and BLHP-B1 samples. (A) The relative molar ratios of monosaccharides among three samples based on the molar of arabinose. (B) HPACE spectrum for monosaccharide detection. All the samples were prepared at concentrations of 1.0 mg/ml.

FIGURE 3

The morphologies of polysaccharides extracted from bamboo leaves. (A,B) Represent BLHP-A1 fraction after being purified by DEAE-Sepharose fast-flow column. (D,E) Illustrate purified BLHP-B1 fraction by DEAE-Sepharose fast-flow column. (C,F) Are the morphologies of BLHP before further purification.

FIGURE 4

FT-IR spectra analysis (A) and UV full scanning (B) of BLHP-A1 and BLHP-B1 fractions.

FIGURE 5

¹H (A) and ¹³C (B) NMR spectra and HSQC for BLHP-A1 and BLHP-B1 samples (C1,C2) after deduction of some weak signal.

FIGURE 6

The measurements of antioxidant activities of BLHP-A1 and BLHP-B1 in chemical view, compared with relevant abilities of VC. (A) DPPH⋅ radicals scavenging of BLHP-A1 and BLHP-B1 samples in the concentration range of 100–1,000 μg/ml. (B) ABTS⁺ radicals scavenging of BLHP-A1 and BLHP-B1 samples in the concentration range of 0.1–1.0 mg/ml. (C) Reducing ability of BLHP-A1 and BLHP-B1 samples. The absorbances were recorded at 700 nm.

FIGURE 7

The effects of BLHP and H₂O₂ concentrations on HepG2 cells viability. (A) The HepG2 cells were incubated with BLHP samples at different concentrations (0, 250, 500, 750, 1,000, 2,000, and 3,000 μg/ml). (B) The HepG2 cells were treated with H₂O₂ at different concentrations (0, 100, 300, 500, 600, 700, 900, 1,100, and 1,300 μmol/ml). (C) The treated concentrations of H₂O₂, BLHP samples in the oxidative stress model. (D) Variations in relative ROS levels compared to that of NC group. The selected concentration of H₂O₂ was 600 μmoL/ml. The selected concentrations of BLHP samples were 250, 1,000 and 3,000 μg/ml. (*p < 0.05, ^**p < 0.01 compared with the control group; #p < 0.05, ##p < 0.01 compared with the H₂O₂ group).

FIGURE 8

The morphologies of HepG2 cells in different situations. (A,B) Were treated with 300 and 600 μmol/ml H₂O₂, respectively. (C) Represented natural cells in MEM without any treatment or protection. (D–F) Illustrated the protection effects in H₂O₂-induced cells by various concentrations (250, 1,000, and 3,000 μg/ml) of BLHP-A1. (G–I) Indicated the protection effects in H₂O₂-induced cells by various concentrations (250, 1,000, and 3,000 μg/ml) of BLHP-B1.

FIGURE 9

The effects of BLHP samples on the concentrations of antioxidant enzymes CAT (A), SOD (B), GSH-Px (C) and MDA (D) product. NC, normal control; H₂O₂: H₂O₂ (600 μmoL/ml) induced group. (*p < 0.05, ^**p < 0.01 compared with the control group; #p < 0.05, ##p < 0.01 compared with the H₂O₂ group).