Structural features, selenization modification, antioxidant and anti-tumor effects of polysaccharides from alfalfa roots

https://doi.org/10.1016/j.ijbiomac.2020.01.239Get rights and content

Highlights

  • Two new polysaccharides were purified from roots of alfalfa.

  • The polysaccharide of alfalfa roots was selenylated for the first time.

  • Selenylated polysaccharide possesses superior antioxidant and anti-tumor activities.

Abstract

Hot water extraction and chromatographic purification methods were used to extract and purify two polysaccharides (RAPS-1 and RAPS-2) from the roots of alfalfa. Subsequently, RAPS-2 was modified using the HNO3/Na2SeO3 method to obtain Se-RAPS-2. The structural features, antioxidant and in vitro anti-tumor activities of the three polysaccharides were evaluated. The structural analysis revealed that RAPS-1 (Mw = 10.0 kDa) was composed of rhamnose, xylose, arabinose, galacturonic acid, mannose and glucose, whereas RAPS-2 (Mw = 15.8 kDa) consisted of rhamnose, xylose, galacturonic acid, mannose, glucose and galactose. RAPS-1 contained 1 → 2, 1 → 4, 1 → 3, and 1 → 6 or 1 → glycosidic bonds; however, while RAPS-2 lacked 1 → 4 glycosidic linkages. The molecular weight of Se-RAPS-2 was 11.0 kDa less than that of RAPS-2. The results of activities demonstrated that Se-RAPS-2 displayed superior antioxidant activity and inhibitory effect in HepG2 cells than RAPS-1 and RAPS-2.

Introduction

Alfalfa (Medicago sativa L.) is a perennial forage crop belonging to the family Fabaceae and genus Medicago; it's widely distributed in Asia, Europe, and Oceania [1]. A mature alfalfa plant can reach the height of 100 cm with a deep, strong taproot and erect, sharply angled leafy stems [2]. Alfalfa is cultivated as an important industrial plant around the world because of its high yield and feeding value [3,4]. Studies suggested have shown that alfalfa stems are rich in polysaccharides with numerous activities, including antioxidant, anti-inflammatory, anti-tumor and lipid-lowering effects. [[4], [5], [6], [7]]. However, because the stem is the only consumable part of the plant, its root is often ignored; therefore, the polysaccharides from alfalfa roots have not been widely studied. Moreover, recent studies on alfalfa polysaccharide have mainly focused on its chemical structures, with few reports on its selenium modification. Thus, it is crucial to investigate the biochemical activities of polysaccharides from alfalfa roots and its selenium modification. This could boost the commercial utility and exploitation of the whole plant.

Selenium (Se) is a trace micronutrient with essential biological functions in the human body [8]. Consuming moderate amounts of Se can increase antioxidant effects and reduce the risk of cancer [9,10]. Compared with inorganic Se compounds, organic Se compounds coupled with proteins or polysaccharides are easily absorbed and exhibit antitumor effect [11,12]. Se-containing polysaccharides include synthesized selenium derivative of polysaccharides and natural selenium polysaccharides extracted from plants, which have stronger bioactivities than non-selenium polysaccharides [13]. Studies have revealed that modification of polysaccharides using selenium could alter their bioactivities and functions, such as antioxidant activity, tumor inhibition effect and anti-inflammatory function [13,14]. Hence, it can be used as an effective method to prepare new products with desirable features.

The objective of this study was to investigate the structural features, antioxidant activities and anti-tumor effects of two novel polysaccharides; RAPS-1 and RAPS-2 isolated from alfalfa roots alongside the selenium modified derivative Se-RAPS-2. This was achieved through the determination of Se content, weight-average molecular mass, identification of polysaccharides using UV, FT-IR, 1D- 2D-NMR methods, and antioxidant activities (ABTS, DPPH radicals-scavenging effects) as well as inhibition effect against HepG2 cells. This study serves as a reference for selenization modification and total utilization of alfalfa plants.

Section snippets

Materials and reagents

Fresh alfalfa plant roots were collected from Chifeng City (Inner Mongolia, China) in September 2016, after identification by Professor Jin-Cai Lu from Shenyang Pharmaceutical University. The voucher specimen (No. 2016-MSL) was kept in the College of Pharmaceutical and Biological Engineering, Shenyang University of Chemical Technology. Neutral protease, AB-8 resin, DEAE-52 cellulose and Sephadex G-200 columns were purchased from Ruida Henghui Co., Ltd. (Beijing, China). Dextrans of different

Purification and selenium modification of polysaccharide

The crude polysaccharide (CP) was separated using a DEAE-52 cellulose column to obtain two major fractions Fr.2 (56.5 mg, 0.17%) and Fr.3 (136.5 mg, 4.1%) with symmetrical elution profiles, which were eluted with 0.2 M and 0.3 M NaCl, respectively. Each fraction was sequentially purified by a Sephadex G-200 column to obtain two purified polysaccharides with single elution peaks, named RAPS-1 and RAPS-2. And their total carbohydrates were 92.5% and 97.1%, respectively (Fig. 1). Subsequently,

Conclusion

In summary, two novel polysaccharides (RAPS-1 and RAPS-2) were successfully isolated from alfalfa roots using DEAE-52 and Sephadex G-200 chromatography columns. Subsequently, a synthesized derivative (Se-RAPS-2) with 320 μg/g of Se was prepared by modifying native RAPS-2 using the HNO3/Na2SeO3 method. Structural analysis revealed that the molecular weights of RAPS-1, RAPS-2, Se-RAPS-2 were 10.0 kDa, 15.8 kDa, and 11.0 kDa, respectively. Unlike the alfalfa polysaccharides reported before, RAPS-1

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Structural features, selenization modification, antioxidant and anti-tumor effects of polysaccharides from alfalfa roots”.

Acknowledgments

This study was supported by the Foundation (no. LQ2019010; L2016009) from the Project of Education Department of Liaoning Province of P. R. China, and the Foundation (no. 20170023) from the Project of Science and Technology Department of Liaoning Province of P. R. China.

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