Preparation, Characterization, and Anti-Adhesive Activity of Sulfate Polysaccharide from Caulerpa lentillifera against Helicobacter pylori

Abstract

In the gastric mucosa, chronic inflammation due to Helicobacter pylori infection promotes gastrocarcinogenesis. Polysaccharides of Caulerpa lentillifera are well-characterized by broad antimicrobial activity and anti-inflammatory potentials. The present study was undertaken to investigate whether the low molecular sulfate polysaccharides of C. lentillifera (CLCP) exhibit any anti-adhesive activity against H. pylori. After a hot water extraction and purification process, two purified polysaccharide fractions (CLCP-1 and CLCP2) were studied based on structural characterization and bioactivity determination. The results implied that except for the molar ratio, CLCP-1 and CLCP-2 contain high sulfate, mannose, galactose, xylose, glucose levels, and low protein levels. The molecular weight and Fourier transform infrared spectroscopy (FT-IR) assays confirmed that CLCP-1 and CLCP-2 are sulfate polysaccharides with an average molecular weight (Mw) of 963.15 and 648.42 kDa, respectively. In addition, CLCP-1 and CLCP-2 exhibited stronger antibacterial activity against H. pylori. CLCP-1 and CLCP-2 could significantly promote macrophage proliferation and decrease the production of nitric oxide (NO) through downregulated expression of inducible nitric oxide synthase (iNOS). Meanwhile, CLCP-1 and CLCP-2 in this study showed efficiently protected gastric adenocarcinoma (AGS) cells against H. pylori with the inhibition of the IL-8/NF-κB axis. These findings suggested the effect of Caulerpa lentillifera polysaccharides on H. pylori adhesion, a potential supply of nutrients for eradication therapy through the reduction of cell count and inflammation.

Keywords: Caulerpa lentillifera; Helicobacter pylori; gastric inflammation; polysaccharide.

Figure 1

Purification of Caulerpa lentillifera polysaccharides fractions using DEAE-Sephadex A-25.

Figure 2

FT-IR spectra (A) and PCA analysis (B) of CLCP, CLCP-1, and CLCP-2.

Figure 3

Antimicrobial activity of CLCP-1 and CLCP-2 against H. pylori ATCC11637 (A), HP43504 (B), HP 51932 (C), and HP 700392 (D). The values are expressed as the means ± SD with * p < 0.05, ** p < 0.01 compared with the control group, n = 3.

Figure 4

Measurement of the potential cytotoxicity of CLCP-1 and CLCP-2 in RAW 264.7 cells (A) an AGS cells (B) by MTT assay. The values are expressed as the means ± SD with ^# p < 0.05 compared with the group without LPS; * p < 0.05 compared with the presence of LPS group, n = 3.

Figure 5

Effect of CLCP-1 and CLCP-2 on NO production (A) and iNOS mRNA expression (B) in LPS-activated RAW 264.7 cells. The values are expressed as the means ± SD with ^## p < 0.05 compared with the control group; * p < 0.05, ** p < 0.01 compared with the LPS group, n = 3.

Figure 6

Inhibitory activity of CLCP-1 and CLCP-2 in H. pylori-infected AGS cells under pre-treatment (A), co-treatment (B), and post-treatment (C). The values are expressed as the means ± SD with * p < 0.05 compared with the control group, n = 3.

Figure 7

CLCP-1 and CLCP-2 inhibit IL-8 and NF-κB mRNA and protein levels in H. pylori-infected AGS cells. (A) IL-8 levels measured by ELISA detection. (B) IL-8 transcription measured by qRT-PCR. (C) NF-κB levels measured by ELISA detection. (D) NF-κB transcription measured by qRT-PCR. ^# p < 0.05 compared with the control group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with the H. pylori group, n = 3.

Figure 7

CLCP-1 and CLCP-2 inhibit IL-8 and NF-κB mRNA and protein levels in H. pylori-infected AGS cells. (A) IL-8 levels measured by ELISA detection. (B) IL-8 transcription measured by qRT-PCR. (C) NF-κB levels measured by ELISA detection. (D) NF-κB transcription measured by qRT-PCR. ^# p < 0.05 compared with the control group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with the H. pylori group, n = 3.