Sulfated modification of arabinogalactans from Larix principis-rupprechtii and their antitumor activities

Highlights

• S-LAGs with different DS were prepared.

• S-LAGs possessed significant antitumor activities in vitro.

• S-LAGs induced apoptosis to inhibit cancer cell proliferation.

Abstract

A highly branched arabinogalactan isolated from Larix principis-rupprechtii and subjected to sulfation derivatization to promote their antitumor bioactivity. Several structural features of the sulfated arabinogalactans (S-LAG) were investigated: molecular weight, monosaccharide constitution, and chemical structures. Spectral analysis indicated that sulfate groups were successfully introduced on arabinogalactan. Sulfated products showed different degrees of substitution (DS) ranging from 0.61 to 0.80, and different Mw ranging from 19.24 to 22.03 kDa. Monosaccharide composition before and after sulfation indicated some level of derivatization selectivity. In vitro cancer cell tests demonstrated that S-LAGs were effective inhibitors to cancer cell growth depending on their dosage. The toxicity mechanisms were further investigated, and assay results revealed that S-LAGs mainly induced cancer cellular apoptosis to promote atrophy and inhibit cell proliferation. The results obtained in this work offer strong demonstration of modified arabinogalactans as a potential medical substance for treating different forms of cancer.

Introduction

Polysaccharides from plants have attracted a great deal of attention because of their biosafety, biological activity and physiological activity (Xie, Jin et al., 2016). Arabinogalactans (AGs) are one particularly interesting class of polysaccharides which can be found in a range of plants such as radish (Shimoda et al., 2014), tamarillo (do Nascimento et al., 2015), green tea (Wang, Shi, Bao, Li, & Wang, 2015), and gum ghatti (Ghosh, Ray, Ghosh, & Ray, 2015). However, there exist uniquely high quantities of AGs in the Larix trees, such as L. occidentalis (Prescott, Groman, & Gulyas, 1997), L. dahurica (Odonmaiig, Ebringerova, Machova, & Alfijldi, 1994), L. laricina (Goellner, Utermoehlen, Kramer, & Classen, 2011), and L. principis-rupprechtii (Tang et al., 2018). Larch arabinogalactan (LAG) is a water-soluble glycan with a high level of molecular branching. It is chemically mainly composed of two monosaccharide residues, d-galactose and l-arabinose, with traces of uronic acid. The inter-residue linkages are founded upon a (1→3)-β-d-galactopyranan main chain with side (1→6)-linked groups of varying length (Goellner et al., 2011; Tang et al., 2018). LAGs have been examined in literature for their immunological activity, their regulatory upon fecal microbial population, and for a variety of ocular benefits (Burgalassi et al., 2007; Currier, Lejtenyi, & Miller, 2003; Grieshop, Flickinger, & Fahey, 2002). However, it does not exhibit possible antitumor activity.

To extend the use of biomass-derived polysaccharides into more application, many studies have been focused on the biological properties of polysaccharide derivatives, especially those which have been sulfated, phosphated, acetylated, or carboxymethylated (Huang & Huang, 2017). In particular, sulfated polysaccharides have been studied extensively due to a variety of reports demonstrating potential health benefits. Sulfated groups change the molecular features of native polysaccharide, changes which significantly influence biological activities (Liu et al., 2015). Some reports on biological properties include demonstration of sulfated polysaccharide's antioxidant (Li, Chi, Yu, Jiang, & Liu, 2017), anticoagulant (Li, Liu et al., 2017) and antiviral activities (Godoi et al., 2014). Furthermore, sulfated polysaccharides were demonstrated as being especially effective at fighting tumors compared to their respective unmodified carbohydrates (Jung, Bae, Lee, & Lee, 2011). Sulfated hyperbranched mushroom polysaccharides were obtained by treatment with chlorosulfonic acid, and the sulfated derivatives exhibited higher antitumor activities against human hepatic cancer cell line (HepG2) (Tao, Zhang, & Cheung, 2006). The sulfated derivatives from Gynostemma pentaphyllum Makino polysaccharide with Mw (8.96 kDa) and DS (1.2) inhibited the growth of HepG2 cells and Hela cells in vitro significantly (Chen et al., 2011). There is no obvious antitumor activity in Radix hedysari polysaccharides, but its sulfated derivatives inhibited proliferation of lung cancer cells and gastric cancer cells by inducing cell apoptosis (Wei, Wei, Cheng, & Zhang, 2012). Recent studies have suggested that sulfated polysaccharides inhibited cancer cell proliferation (Wang, Zhang, Yu, & Cheung, 2009). This cytostatic effect may be due to the induction of apoptosis (Zhong et al., 2015). A large number of new drugs and nutraceuticals based on the bioactivities of sulfated polysaccharides are currently under development (Scott & Alyssa, 2013). Therefore, the development of more types of sulfated polysaccharides is gaining more attention.

Methods for sulfation of polysaccharides includes the chlorosulfonic acid-pyridine (CSA-Pyr) method, SO₃-pyridine (SO₃-Pyr) method and concentrated sulfuric acid method. Among them, CSA-Pyr method has been elucidated as superior over recent years due to its advantageously high yield, degree of substitution (DS), and convenient handling (Raveendran, Yoshida, Maekawa, & Kumar, 2013). It was reported that DS and Mw are the critical parameters influencing the potency of biological activities exhibited by sulfated polysaccharides (Wang, Li, & Chen, 2009). Mw depends on the structure of the glycan itself as well as the methods of sulfation. Different methods can cause cleavage at various points of polysaccharide molecules, and the extent of cleavage is shown to wildly vary using the same modification method across different polysaccharides. Li, Chi et al. (2017) reveal that sulfated derivatives of the two different Mw polysaccharides were synthesized by CSA-Pyr method, and the DS of SPEP and SLEP were 0.57 and 0.81, respectively. In another work, the polysaccharides from the leaves of C. paliurus were subjected to sulfation with SO₃-Pyr complex, producing the sulfated derivatives with different DS (0.12–0.55) (Xie, Jin et al., 2016). Therefore, a variety of modification methods provide a way to prepare a family of new polysaccharide derivatives with different structural characteristics, which can be used for structure/property studies.

In our previous research, we successfully isolated and purified an arabinogalactan from L. principis-rupprechtii. Starting from this same material, in this work we have performed sulfation on LAG understanding that this sort of work has not yet been performed. Furthermore, we sought to advance the state of knowledge concerning the structure-function relationship between sulfated polysaccharides and antitumor activity. For this reason, four sulfated arabinogalactans (S-LAGs), with various DS, Mw and carbohydrate contents, were prepared using the CSA-Pyr method. The chemical structures of the S-LAGs were next exhaustively characterized using a variety of techniques to establish a baseline understanding of their chemical structure and inter-sample differences. Furthermore, antitumor activities of S-LAGs were investigated by CCK-8 assays. Any relationship between the obtained assay responses and the structural properties of S-LAGs were identified. The intent behind this work was two-fold: to further develop the value of LAGs and specifically S-LAGs, while attempting to further the understanding of which S-LAG properties most improve antitumor activities.