molecules Article Isolation, Purification and Structural Characterization of Two Novel Water-Soluble Polysaccharides from Anredera cordifolia Zhi-Peng Zhang 1,2, Can-Can Shen 1, Fu-Li Gao 1, Hui Wei 1, Di-Feng Ren 1,* ID and Jun Lu 2,* 1 Beijing Key Laboratory of Forest Food Processing and Safety, College of Biological Sciences and Biotechnology, Beijing Forestry University, 100083 Beijing, China; [email protected] (Z.-P.Z.); [email protected] (C.-C.S.); [email protected] (F.-L.G.); [email protected] (H.W.) 2 Beijing Engineering Research Center of Protein & Functional Peptides, China National Research Institute of Food & Fermentation Industries, 100015 Beijing, China * Correspondence: [email protected] (D.-F.R.); [email protected] (J.L.); Tel.: +86-10-6233-6700 (D.-F.R.); +86-10-5321-8278 (J.L.); Fax: +86-10-6233-8221 (D.-F.R.); +86-10-5321-8278 (J.L.) Academic Editor: Quan-Bin Han Received: 5 July 2017; Accepted: 29 July 2017; Published: 3 August 2017 Abstract: Anredera cordifolia, a climber and member of the Basellaceae family, has long been a traditional medicine used for the treatment of hyperglycemia in China. Two water-soluble polysaccharides, ACP1-1 and ACP2-1, were isolated from A. cordifolia seeds by hot water extraction. The two fractions, ACP1-1 and ACP2-1 with molecular weights of 46.78 kDa ± 0.03 and 586.8 kDa ± 0.05, respectively, were purified by chromatography. ACP1-1 contained mannose, glucose, galactose in a molar ratio of 1.08:4.65:1.75, whereas ACP2-1 contained arabinose, ribose, galactose, glucose, mannose in a molar ratio of 0.9:0.4:0.5:1.2:0.9. Based on methylation analysis, ultraviolet and Fourier transform-infrared spectroscopy, and periodate oxidation the main backbone chain of ACP1-1 contained (1!3,6)-galacturonopyranosyl residues interspersed with (1!4)-residues and (1!3)-mannopyranosyl residues. The main backbone chain of ACP2-1 contained (1!3)-galacturonopyranosyl residues interspersed with (1!4)-glucopyranosyl residues. Keywords: isolation; purification; polysaccharide; Anredera cordifolia; structural characterization 1. Introduction Polysaccharides, which are made up of monosaccharides linked through glycosidic bonds, exist in plants, animals, and microorganisms. In recent years, some bioactive polysaccharides extracted and separated from plants, especially from medicinal plants, have attracted significant attention in the fields of pharmacology and biochemistry because of their potential biological activities, such as antioxidant [1,2], α-glucosidase inhibitory [3], anticoagulant [4,5], prebiotic [6], antitumor [7] and immunobiological effects [8]. Most polysaccharides derived from plants are relatively nontoxic, and any side effects are minimal compared with those of synthetic compounds [9,10]. Thus, increasing attention has been devoted to the study of bioactive polysaccharides and most of these studies performed on bioactive polysaccharides took traditional information on the use of medicinal plants and their related bioactivities as a guide to choose the plants that should be studied [11–13]. Meanwhile, many novel techniques such as ultrasound assisted extraction, ultrafiltration, centrifugation and pulsed electric energy have been applied to the extraction and purification of bioactive polysaccharides [14–16]. The search for efficient methods of extraction and purification of bioactive polysaccharides from plant has thus become a research hotspot. Molecules 2017, 22, 1276; doi:10.3390/molecules22081276 www.mdpi.com/journal/molecules Molecules 2017, 22, 1276 2 of 13 Anredera cordifolia is a climbing succulent that is widely distributed in the southern parts of South America and China [17]. It belongs to the family Basellaceae, which includes 19 accepted species in four genera. In daily life, the tubers and leaves are used in traditional medicine applications as a sedative, supplement, and for the treatment of hyperglycemia [18]. In previous studies, A. cordifolia was reported to contain bioactive compounds that possess antibacterial, anti-obesity and anti-hypoglycemic, cytotoxic and anti-mutagenic, anti-diabetic, and anti-inflammatory activities [19]. Polysaccharides are found to be primary active components from many medicinal plants such as Dendrobium plants, Astragalus membranaceus and Dimocarpus longan Lour, which exert various pharmacological activities [20]. However, current studies have focused mainly on educts and their biological activities, and few reports describe the structural characteristics of polysaccharides extracted from A. cordifolia. The biological activities of polysaccharides are correlated with their structural characterization. The type of monomer, linkage type and position, and the number and position of branches occurring within the polymer chain strongly influence the three-dimensional arrangement, and in addition to the molecular size, these factors determine polysaccharide behavior [21]. Physical properties, such as solubility, viscosity, and gelation, may also influence the biological activity because they can affect bioavailability [22,23]. Some studies suggested that polysaccharides have significantly different average molecular weights, however, fractions with similar monosaccharide compositions can display the same biological activity [24]. Therefore, the elucidation of molecular structures of polysaccharides occurring in medicinal plants is very important for predicting their biological behavior. To our knowledge, there are no reports available on the structural elucidation of polysaccharides from A. cordifolia. Hence, the present study aimed to separate, purify, and structurally characterize water-soluble polysaccharides from A. cordifolia. The results could be used for further investigation of the structure-activity relationship and development of applications of A. cordifolia polysaccharides. 2. Results and Discussion 2.1. Isolation and Purification of Polysaccharides The crude polysaccharide ACP was obtained as a water-soluble white powder from A. cordifolia by hot-water extraction, ethanol precipitation, and deproteinization by the Sevag method, trichloroacetic acid method, and hydrochloric acid method. Decolorization was performed using five different macroporous resins. Among the three methods tested, the trichloroacetic acid method was the most effective approach to eliminate the protein, giving the highest protein clearance rate and polysaccharide retention rate among the tested methods. The hydrochloric acid method may cause partial hydrolysis of polysaccharides, resulting in reduced polysaccharide retention rate [25]. Other studies have reported that the efficiency of the Sevag method for the removal of proteins from Ganoderma sinensis was significantly lower compared with that of the trichloroacetic acid method, which is consistent with our results [26]. The protein clearance rate with the Sevag method was similar to that with the trichloroacetic acid method, but had only half of the polysaccharide retention rate of the trichloroacetic acid method (Table1). There was a significant loss in the recovery of polysaccharides, which may be due to damage to the polysaccharides by the Sevag reagent as well as the co-removing effects of the glucoprotein during the harsh chemical treatment procedure [27]. As shown in Table2, X-5 resins exhibited higher polysaccharide retention rates and decolorization rates than those of the other four resins. The low retention rates of polysaccharide on AB-8, D-101, HPD-400 and NKA-9 resins were probably a result of the high molecular weight and the high space structure of polysaccharides [28]. After deproteinizing and decolorizing, the polysaccharide content was 25.36%. The collected residues contained trace amounts of proteins and pigments. After submitting to condensation, precipitation, dialysis with water, and freeze-drying, ACP was further purified on the DEAE-cellulose A52 and Sephadex G-100 gel-filtration columns (Figure1). The main fractions were collected, lyophilized, and named as ACP1-1 and ACP2-1 for further structural characterization. Molecules 2017, 22, 1276 3 of 13 Molecules 2017, 22, 1276 3 of 12 Table 1. Deproteinization effects by different methods in ACP. Table 1. Deproteinization effects by different methods in ACP. Effect Trichloroacetic Acid Sevag Hydrochloric Acid Effect Trichloroacetic Acid Sevag Hydrochloric Acid Protein clearance rate/% 97.73 ± 0.52 97.31 ± 0.56 55.10 ± 0.43 Protein clearance rate/% 97.73 ± 0.52 97.31 ± 0.56 55.10 ± 0.43 Polysaccharide loss rate/% 4.86 ± 0.37 49.67 ± 0.26 13.53 ± 0.35 Polysaccharide loss rate/% 4.86 ± 0.37 49.67 ± 0.26 13.53 ± 0.35 Table 2. Decolorization effects by different methods in ACP. Table 2. Decolorization effects by different methods in ACP. ResinResin PolarityPolarity Decolorization Decolorization Rate Rate/%/% Polysaccharide Polysaccharide Retention Retention Rate Rate/%/% D101D101 ApolarApolar 55.24 ± 0.240.24 59.19 59.19 ± ±0.440.44 X-5X-5 ApolarApolar 72.16 ± 0.470.47 75.24 75.24 ± ±0.170.17 AB-8AB-8 WeekWeek polar polar 59.13 ± 0.320.32 54.43 54.43 ± ±0.290.29 HPD-400HPD-400 WeekWeek polar polar 43.86 ± 0.310.31 65.76 65.76 ± ±0.310.31 NKA-9NKA-9 PolarPolar 45.59 ± 0.280.28 68.38 68.38 ± ±0.230.23 Figure 1. Elution profiles profiles of crude polysaccharide ACP on aa DEAE-52DEAE-52 column.column. 2.2. Homogeneity and Molecular Weights ofof ACP1-1ACP1-1 andand ACP2-1ACP2-1 The polysaccharides ACP1-1 and ACP2-1 each showed only one symmetrical and narrow peak withwith anan elutionelution time time of of about about 21 21 min min in thein