Assessment of biochemical and immunomodulatory activity of sulphated polysaccharides from Ulva intestinalis

Highlights

• Sulphated polysaccharides from U. intestinalis fractionated on a silica-silica column activated macrophages.

• The molecular weight and sulphate/total sugar ratio affect the immunomodulatory activity of sulphated polysaccharides.

• Fractionated sulphated polysaccharides showed higher scavenging activity and immunomodulatory activity than unfractionated sulphated polysaccharides.

Abstract

The biochemical characteristics and immunomodulatory activity of sulphated polysaccharides isolated from Ulva intestinalis and fractionated using a silica-silica column were investigated. The unfractionated (USP) and fractionated sulphated polysaccharides (FSP4, FSP30, and FSP32) consisted mostly of carbohydrates (4.84–26.55%) and sulphates (2.85–20.42%). Structural analyses showed that USP, FSP4, FSP30 and FSP32 had molecular weights of 300, 80, 110 and 140 kDa, respectively. FSP30 exhibited the strongest DPPH radical scavenging activity. Moreover, FSP30 showed stronger immunomodulatory activities than UPS in term of stimulating the production of pro-inflammatory cytokines, including nitric oxide (NO), tumour necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), in macrophage J774A.1 cells. USP and FSP30 were not cytotoxic to mouse macrophage at the tested concentrations (6.25–50 μg/mL). The results suggested that U. intestinalis polysaccharides could be explored as potential antioxidant and immunomodulatory agents to be used as complementary medicine or functional foods.

Introduction

Sulphated polysaccharides are bioactive macromolecules in which some of the hydroxyl groups of the sugar residues are substituted by sulphate groups. These anionic polysaccharides are ubiquitous in nature and occur in a wide variety of organisms, including mammals, invertebrates and flora [1]. In algae, sulphated polysaccharides are the major component of the cell wall matrix [2]. Sulphated polysaccharides from marine algae have been mainly found in brown algae (Phaeophyta), such as fucoidans, ascophyllan, sargassum and glucuronoxylofucan, and in red algae (Rhodophyta), such as agar and carrageenan [1]. In contrast to sulphated polysaccharides found in brown and red algae, the major sulphated polysaccharide of green algae is more heterogeneous in sugar composition; its three main groups are glucuronoxylorhamnans, glucuronoxylorhamnogalactans and xyloarabinogalactans [3]. These sulphated polysaccharides are of particular interest because they are bioactive, i.e., they exert anticoagulant, antiviral, antitumor and anti-inflammatory activity [4].

Ulva intestinalis is a green seaweed belonging to the family Ulvaceae. They are a rich source of sulphated polysaccharides that are referred to as ulvans. Sulphated polysaccharides mainly contain the sugars rhamnose, xylose, and glucuronic acid, as well as small amounts of other sugars (galactose, arabinose, mannose, and glucose) [5]. Sulphated polysaccharides from U. intestinalis feature a typical sugar backbone, like glucose and rhamnose, and sulphate is attached to rhamnose at C-2 or C-3 [6]. Various monosaccharide components of the sulphated polysaccharide chain, such as l-arabinose, d-glucose, l-mannose and l-rhamnose, promote antioxidant [7] and immunomodulatory activity [8]. Specifically, l-rhamnose and α-l-rhamnose oligosaccharides have been described to strongly interact with specific receptors on several proteins that are involved in immunomodulatory activity [7].

The α-l-rhamnose, a component of glycosides or polysaccharides, is especially distributed in plants and microorganisms and recognized by functional antibodies in humans, which is an important aspect of immunomodulatory activity [9]. Sulphated polysaccharides also exert various biological and pharmacological activities, including immunomodulatory activity [1]. The immunomodulatory activities of sulphated polysaccharides depend on their characteristic parameters, such as the degree of sulphation (DS), carbohydrate content and sulphate content/total sugar ratio [10].

Sulphated polysaccharides from algae have been reported to stimulate the secretory activity of macrophages, induce the production of nitric oxide (NO), and enhance the secretion of cytokines and chemokines, such as tumour necrosis factor (TNF-α) and interleukin IL-1β [11]. Leiro et al. [12] reported that sulphated polysaccharides from Ulva rigida extracted with distilled water and purified by anion exchange were potent immunomodulators, stimulating murine macrophage RAW 264.7 cells and inducing the secretion of nitric oxide (NO). In a previous study, sulphated polysaccharides from Codium fragile extracted with a solvent enhanced the production of pro-inflammatory cytokines from RAW 264.7 cells, including interleukins-1, 6, and 12, tumour necrosis factor-α and anti-inflammatory (IL-10) cytokines [13]. Furthermore, RAW 264.7 cells stimulated with fractionated sulphated polysaccharides from red algae exhibited the increases in TNF-α production than cells stimulated with lipopolysaccharides (LPS), the endotoxin from gram-negative bacteria, which could be elicited a variety of responses in macrophages, including stimulating nitric oxide production [2]. Additionally evidence suggests that sulphated polysaccharides extracted from Capsosiphon fulvescens and polysaccharides fractionated by ion-exchange chromatography might be strong stimulators of RAW264.7 cells, resulting in the production of a considerable amount of NO [14]. According to Paul et al. [15], protein kinase C activation has been identified as an early response in LPS-stimulated macrophages and might be essential for the up-regulation of NO production. Furthermore, the fraction of sulphated polysaccharides separated by DEAE-Sepharose from Prunella vulgaris L. showed stronger immunomodulatory activities than crude sulphated polysaccharides [16].

Although the previous study have explored the antioxidant activity of hot water-extracted sulphated polysaccharides from U. intestinalis [6], the research on the biochemical and immunomodulatory activity of U. intestinalis polysaccharides fractionated with a silica-silica column has not been reported. The present study was designed to explore the immunomodulatory activity of sulphated polysaccharides and fractionated sulphated polysaccharides from U. intestinalis. The effects of sulphated polysaccharide fractions on macrophage viability, nitric oxide production, and cytokine production were also monitored in order to explore the immunomodulatory activity and induce transcription of key cytokines for further useful immunopotentiators.