**1. Introduction**

Purslane (*Portulaca oleracea* L.) is a wild plant belonging to the *Portulacaceae* family. Purslane is widely spread and popular in most areas, including China, Europe, and Mediterranean countries, and is edible but usually regarded as one of these non-conventional plants. More importantly, purslane is regarded as having many biofunctions in both medicine and food fields. It was first recorded in the Compendium of Material Medica that purslane leaves had the ability to clear evil heat and remove toxins [1]. Moreover, recent research results have indicated the emerging functional properties of purslane in the intestine, skin, nerve, respiratory, and other systems. For example, the extract of purslane leaves showed

**Citation:** Lin, Y.-R.; Guan, Q.-Y.; Li, L.-Y.; Tang, Z.-M.; Zhang, Q.; Zhao, X.-H. In Vitro Immuno-Modulatory Potentials of Purslane (*Portulaca oleracea* L.) Polysaccharides with a Chemical Selenylation. *Foods* **2022**, *11*, 14. https://doi.org/10.3390/ foods11010014

Academic Editors: Jianhua Xie, Yanjun Zhang and Hansong Yu

Received: 18 November 2021 Accepted: 18 December 2021 Published: 21 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

an ability to reduce the severity of colitis through regulating the immune mechanism involved in the pathogenesis of colitis [2]. Besides, it was also found that the purslane juice could protect the rat brain from the rotenone-caused neurotoxicity, as well as apoptosis, by inhibiting excessive oxidative stress [3]. Overall, purslane is considered to contain these bioactive components, including alkaloids, polysaccharides, unsaturated fatty acids, flavonoids, proteins, and others, thus being regarded with various beneficial functions, such as anti-bacterial, anti-fungal, anti-inflammatory, analgesic, muscle relaxant, and wound healing effects. In addition, purslane is unusually rich in aliphatic acids (e.g., the α-linolenic acid) that are important in cholesterol reduction and blood lipid-lowering, as well as antithrombotic or anti-cardiovascular effect [4], while the flavone compounds in purslane also are of importance for the vital hypoglycemic and anti-oxidative functions by inhibiting the Akt phosphorylation to enhance the consumption of glucose or by scavenging free radicals and reducing metal ions, such as Fe3+ [5]. Overall, the potential health benefits of purslane are still insufficiently investigated so far.

Polysaccharides, a kind of carbohydrates, are made up of more than ten monosaccharide units that are joined through the glycosidic bonds in the branched or unbranched chains. For purslane, it was reported the polysaccharides extracted by water had a molecular mass about 7.3 kDa, with arabinose, galactose, glucose, mannose, rhamnose, and xylose as main saccharide elements [6]. It is worth mentioning that natural polysaccharides have various pharmacological effects, such as anti-cancer, anti-inflammation, anti-oxidation, modulation of gut microbiota, and immune function [7]. For example, the natural polysaccharides might exert hepatoprotective effect by regulating the pathways of inflammation and apoptosis, lipid metabolism, and cytochrome P450 enzymes [8], while the acidic polysaccharides from *Schisandra chinensis*, through reducing the oxidative stress, could protect the acute liver injury induced by ethanol [9]. Additionally, the combined fungal polysaccharides could suppress the hepatotoxicity induced by cyclophosphamide through reducing toxicity markers and preventing inflammatory responses [10]. As is reported, anti-oxidation and hypoglycemic effect are two important biological functions of plant polysaccharides. It was reported that the polysaccharides from garlic (*Allium sativum* L.) bolt and green walnut (*Juglandaceae*) husk possessed anti-oxidant activity to scavenge three radicals or reduce the multi-valent metal ions, such as Fe3 <sup>+</sup> [11,12], while those from bluish dogbane (*Apocynum venetum*) leaves had anti-hypoglycemic effect in type 2 diabetes mice by regulating intestinal flora, along with reducing glucose absorption [13]. Today, cancer is one of the most fatal diseases in the world; thus, the anti-tumor activities of polysaccharides by inhibiting tumor growth and enhancing immunological functions have attracted a special attention in recent [14,15]. For example, the polysaccharides from shiitake mushrooms (*Lentinus edodes*) could exert anti-tumor activities to the colon cancer HT-29 cells via cell proliferation suppression and apoptosis induction, through an internal pathway mediated by reactive oxygen species (ROS) and external pathway engaged with TNF-α [16]. In referring purslane polysaccharides, they were reported to have anti-diabetic effect in diabetic rats and could enhance the immune state of the rats with gastric cancer [6,17].

Immune modulation of natural plant polysaccharides and other components are also sufficiently studied. The immune system is the body's defensive system that performs the immune responses, immune function, and self-protection, and is composed of these elements, such as immune organs, cells, and molecules. The immunological responses consist of the innate and adaptive immune responses, including humoral and cellular immunity, while the responses to external stimulation are considered as one of the body's key defending strategies to prevent and combat external infections, inflammation, and cancers [18]. Some natural substances derived from natural foods have immuno-modulatory effects. Tea polyphenols could increase the immunity of tilapia via promoting the activity and expression of immunoglobulin, enhancing the lysozyme activity, and regulating the NF-κB signaling pathway [19]. The peptides derived from whey and casein proteins had a terrific immuno-modulatory function because the peptides could increase the macrophage phagocytosis, promote splenocyte proliferation, and enhance cytokine secretion [20,21]. In

addition, a flavonoid compound, quercetin, also might improve the immunity of *Arbor Acre* broilers [22]. Overall, it was revealed that plant polysaccharides could play an effective immuno-modulatory role in immune systems through activating the macrophages, splenocytes, and other immune cells, promoting the release of cytokines, increasing the growth of immune organs and the secretion of immunoglobulins, and inhibiting the overactivation of the complement system [23]. However, whether a chemical modification of natural polysaccharides will cause positive or negative effects on the immune modulation of the modified polysaccharides is less studied in the present. Thus, such an investigation using the soluble purslane polysaccharides as a target for a chemical selenylation deserves our consideration.

In this study, the soluble polysaccharides from purslane (namely PSPO) were extracted by water at a neutral condition, and then selenylated chemically using the Na2SeO3-HNO3 system for two selenylation extents to prepare two selenylated PSPO products (SePSPO), namely SePSPO-1 and SePSPO-2, respectively. Both SePSPO-1 and SePSPO-2 were assessed for their in vitro immuno-modulatory activities using two immune cells (i.e., the RAW 264.7 macrophages and murine splenocytes) as cell models and the unmodified PSPO as a control. Several indices, such as growth proliferation, phagocytic activity, cellular secretion of five cytokines, and T lymphocyte subpopulations, were measured and compared to reflect the target immune modulation. The purpose of this study was to disclose whether the performed chemical selenylation could cause bioactivity changes for the soluble PSPO in their important immune potential.

#### **2. Materials and Methods**
