Structure characterization of two novel polysaccharides from Colocasia esculenta (taro) and a comparative study of their immunomodulatory activities

Journal of Functional Foods 42 (2018) 47–57

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Structure characterization of two novel polysaccharides from Colocasia esculenta (taro) and a comparative study of their immunomodulatory activities ⁎

Huixian Li, Zhou Dong, Xiaojia Liu, Huamin Chen, Furao Lai , Mengmeng Zhang

T

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College of Food Sciences and Engineering, South China University of Technology, Guangzhou, Guangdong 510640, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Non-starch polysaccharides Colocasia esculenta Structural characterization Immunomodulatory activity

Two fractions of non-starch polysaccharides termed TPS-1 and TPS-2 were sequentially purified from the corms of Colocasia esculenta. Structural characterizations of TPS-1 and TPS-2 were determined. The average molecular weight of TPS-1 and TPS-2 were estimated to be 10.502 and 10.191 kDa, respectively. TPS-1 consisted of Rha (8.5%), Xyl (0.73%), Glc (88.57%), and Gal (2.56%), while TPS-2 consisted of Rha (3.54%), Arf (1.6%), Glc (87.92%), and Gal (6.94%). The presence of glycosidic linkages in the two fractions of polysaccharides were identified and were significantly different. TPS-2 exhibited a higher immunomodulatory activity than TPS-1 through an immunostimulation assay promoting NO, TNF-α, and IL-6 production in RAW 264.7 cells. Furthermore, TPS-2 could significantly enhance the phagocytosis ability of macrophages. The membrane receptors of TPS-2 were identified to be TLR2, TLR4, GR, and SR. Taken together, TPS-1 and TPS-2 could be considered as novel potential immunostimulants of food and pharmaceutical supplements for hypoimmunity.

1. Introduction Colocasia esculenta, commonly known as taro, is an edible plant that is widely distributed in China, Japan, Korea, and other subtropical or tropical areas. The carbohydrate content of taro is high, indicating that taro is a good source of energy (Kaur, Kaushal, & Sandhu, 2013). In recent decades, various active components, including resistant starch, mucilage, anthocyanins, hemagglutinin, non-starch polysaccharides, protein, tarin, lectin etc., have been identified in taro (Chan, Wong, & Ng, 2010; Ghan, Kao-Jao, & Nakayama, 1977; Jiang, Ramsden, & Corke, 1997; Kundu et al., 2012; Liu, Donner, Yin, Huang, & Fan, 2006; Njintang et al., 2014; Park, Lee, Cho, Kim, & Shin, 2013). Such compounds have been proven to possess effective slowly digested antitumor, anti-metastatic, antioxidative, and anti-inflammatory abilities (Cambie & Ferguson, 2003; Chan et al., 2010; Kundu et al., 2012; Park et al., 2013). For example, lectin, a kind of glycoprotein, is considered to be involved in the regulation of different cellular activities, including the effect of lectins as cytokine-mimetic molecules to modulate innate and adaptive immune responses under physiological or pathological conditions (Pereira et al., 2014; Pereira, Silva, Verícimo, Paschoalin, & Teixeira, 2015; Toscano et al., 2007). These characteristics and nutritional benefits of Colocasia esculenta make it suitable for wide consumption. Previously, much attention has been focused on the starch

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content of taro. In contrast, limited studies have been conducted on the non-starch polysaccharides of taro. To date, a couple of polysaccharides have been purified from Colocasia esculenta, but little is known about their chemical structure and immunomodulatory activities (Jiang et al., 1997; Park et al., 2013). Especially, the presence of glycosidic linkages in taro non-starch polysaccharides is still unknown, and only their antimetastatic activity has been reported (Park et al., 2013). The more detailed elucidation of their chemical structures and immunomodulatory activities need further exploration. Besides, the relationship between their structures and activities need be investigated and discussed to explain their observed bioactivity. More interestingly, while most of the raw materials used in previous studies were small cultivar of taro, our research is based on a large cultivar of taro (Lechang, Shaoguan, China) with a weight and length of more than 2.5 kg and 30 cm per corm, respectively. However, little research about this kind of taro exists, and attention should be placed on this kind of large taro to explore and complement the effectiveness of this vegetable. Macrophages, which fight against inflammation and infection, play an essential role in both innate and adaptive immune responses (Cassetta, Cassol, & Poli, 2011). They can act as antigen-presenting cells, which perform immune functions such as phagocytosis, surveillance, chemotaxis, and the destruction of harmful substances, suggesting that their activation helps to regulate immunity (Xie et al.,

Corresponding authors at: Department of Food Quality and Safety, South China University of Technology, Wushan Road 381, Guangzhou, Guangdong, China. E-mail addresses: [email protected] (F. Lai), [email protected] (M. Zhang).

https://doi.org/10.1016/j.jff.2017.12.067 Received 29 June 2017; Received in revised form 25 December 2017; Accepted 29 December 2017 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.

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dialysis tubes (molecular weight cutoff = 3500 Da), and then concentrated and lyophilized. The crude polysaccharides (100 mg) were dissolved in ultrapure water and injected into a DEAE Sepharose Fast Flow anion-exchange chromatography column (1.6 × 20 cm), equilibrated with a step gradient from 0 to 0.5 M NaCl solutions at a flow rate of 1.0 mL/min. The eluent was collected and analyzed by the phenol–sulfuric acid method (DuBois, Gilles, Hamilton, Rebers, & Smith, 1956). Subsequently, the two fractions, TPS-1 (eluted by distilled water) and TPS-2 (eluted by 0.1 M NaCl), were obtained. The eluents of TPS-1 or TPS-2 were dialyzed against ultrapure water for 48 h, followed by freeze-drying. TPS-1 and TPS-2 were further purified using a Sephacryl S-200 High Resolution column. A 20 mg sample was re-dissolved in 4 mL of deionized water and loaded onto a column of Sephacryl S-200 High Resolution (1.2 × 100.0 cm). The column was eluted by ultrapure water at a flow rate of 0.3 mL/min. The elution was monitored using the phenol–sulfuric acid method. After collecting and concentrating the eluate, the resulting polysaccharide solution was dialyzed and lyophilized. The carbohydrate contents of TPS-1 and TPS-2 were 96.5 and 97.2%, respectively, as analyzed by the phenol–sulfuric acid method with D-glucose as a standard.

2016). A number of studies have confirmed that activated macrophages might exert immune responses by phagocytosis, releasing corresponding effective molecules and cytokines such as NO, IL-6, and TNF-α (Cheng, Wan, Wang, Jin, & Xu, 2008; Deng et al., 2016; Liu et al., 2006). Therefore, macrophages are commonly regarded as an ideal cell model to evaluate the immunomodulatory effects of polysaccharides. In the present study, two fractions of new polysaccharides, termed TPS-1 and TPS-2, were purified from the corm of Colocasia esculenta. Their chemical structures were characterized by investigating their molecular weights, conformation, monosaccharide compositions, and glycosidic bonds. Meanwhile, a comparative effect of the two polysaccharides on stimulating the secretion of cytokines NO, IL-6, and TNF-α by RAW 264.7 cells was investigated to discuss their structure–activity relationship. In addition, the phagocytic capacity of TPS-2 was determined to further explore its immunomodulatory activities. Finally, we determined the membrane receptors of TPS-2 on RAW 264.7 cells to find out the potential mechanisms of its signaling pathway. 2. Materials and methods 2.1. Material and chemicals

2.3. Structural characterization of polysaccharide TPS-1 and TPS-2 The corms of Colocasia esculenta were obtained from Lechang, Shaoguan, China. Diethylaminoethyl (DEAE)-Sepharose Fast Flow and Sephacryl S-200 High Resolution were acquired from Shanghai Yuanye Bio-Technology Company Limited (Shanghai, China). Myoinositol, as well as the standards of phycite, glycerol, and glycol and monosaccharide references (e.g., arabinose, glucuronic acid, glucose, galactose, xylose, mannose, fucose, and rhamnose) were purchased from Sigma Company (St. Louis, MO, USA). The murine macrophage cell line RAW 264.7 was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The Dulbecco’s modified Eagle’s medium (DMEM) culture medium, fetal bovine serum (FBS), phosphate-buffered saline (PBS, pH 7.4), streptomycin, and penicillin were obtained from Gibco Life Technologies (Grand Island, NY). Lipopolysaccharide (LPS) was acquired from Sigma Company (St. Louis, MO, USA). The Vybrant Phagocytosis Assay Kit was purchased from Thermo Fisher Scientific (USA). Antibodies, such as the anti-scavenger receptor (anti-SR) I antibody, anti-mannose receptor (anti-MR) antibody, anti-beta glucan receptor (anti-GR) antibody, anti-toll-like 2 (anti-TLR2) antibody, anti-complement receptor 3 (anti-CR3) antibody, and anti-toll-like receptor 4 (anti-TLR4) antibody, were obtained from Abcam, Inc. (Cambridge, MA). The mouse IL-6 and TNF-α ELISA kit were purchased from R&D Systems, Minneapolis, USA. The Griess reagent was obtained from Sigma-Aldrich (Australia). All chemical reagents used in this study were of analytical grade.

2.3.1. Molecular weight determination The molecular weight of TPS-1 and TPS-2 were determined using high-performance gel permeation chromatography (HPGPC). A TSK G5000PWXL column (7.8 × 300 mm), TSK G-3000PWXL column (7.8 × 300 mm) and Waters 2414 differential refractive index detector were applied to HPGPC. Each sample (3 mg/mL) was eluted by 0.02 M KH2PO4 at a flow rate of 0.6 mL/min. 2.3.2. Infrared spectral analysis Three milligrams of TPS-1 and TPS-2 samples were ground to a fine powder and detected on a Fourier transform infrared (FT-IR) spectrometer (Bruker, Germany) in the 4000–400 cm−1 region. 2.3.3. Determination of triple-helix structure The conformational structures of the two polysaccharides were determined according to the Congo red test (Wang et al., 2013). 2.3.4. Analysis of monosaccharide composition TPS-1 or TPS-2 (10 mg) was hydrolyzed with 4 mL of trifluoroacetic acid (TFA, 2 M) at 110 °C for 5 h in a sealed ampoule. In order to remove the redundant TFA, the solution was completely dried by rotary evaporation, and the resulting dry sample was redissolved in methanol followed by further evaporation to dryness. Subsequently, the acidolysis products were mixed with hydroxylamine hydrochloride and pyridine (1 mL) at 90 °C for 30 min. Thereafter, 1 mL of acetic anhydride was added to the resultant solution, which was continuously heated for another 30 min. After being passed through a 0.22-μm nylon membrane, the filtrate was analyzed using a gas chromatograph (GC) (Agilent, USA) equipped with a HP-5 capillary column (30 m × 320 μm × 0.25 μm, Agilent) and a flame ionization detector. The flow rate of N2 was 20.0 mL/min, and the temperature of the column was increased from 150 to 200 °C at the rate of 2.5 °C/min, and then maintained for 12 min. A series of monosaccharides (arabinose, glucuronic acid, glucose, galactose, xylose, mannose, fucose, and rhamnose) were used as standards. Myoinositol was used as an internal standard.

2.2. Extraction and purification of polysaccharides from the corms of Colocasia esculenta The dry corms of Colocasia esculenta (taro) were crushed to powder using a tissue triturator. The root powder was mixed with ultrapure water at a ratio of 1:30 (w/v). After a microwave treatment at 400 W for 80 s, the corm powder was extracted twice with 80 °C water, each time for 3 h. The water extract was combined and centrifuged at 4000g for 15 min. After the supernatant was collected and concentrated at 55 °C, α-amylase and glucoamylase were applied to remove the starch until the iodine reaction led to the disappearance of the blue color, indicating the complete removal of starch, as reported previously (Wang et al., 2016). The deproteinization process was performed 12 times using the Sevag method (Staob, 1965). Four volumes of 95% ethanol were added to the resultant solution and it was incubated at 4 °C overnight. Subsequently, this solution was centrifuged at 2500g for 10 min, and the resulting precipitate was redissolved in distilled water. The crude polysaccharides were dialyzed against distilled water using

2.3.5. Periodate oxidation-smith degradation A total of 25 mg of TPS-1 or TPS-2 was dissolved in 12.5 mL of distilled water and mixed with 12.5 mL of NaIO4 (30 mM) at 25 °C in the dark. At defined intervals (0, 6, 12, 24, 36, and 48 h), 0.1 mL of the reaction liquid was taken out from the mixture to detect the absorbance 48

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incubated for 6 h. Then, an FITC-labeled Escherichia coli solution from the Vybrant Phagocytosis Assay Kit was added to the resulting cells and incubated for 2 h. After the suspension was removed from the cells, a total of 100 μL trypan blue solution was added to the culture plate and incubated for 1 min. Subsequently, the rest of the trypan blue was removed as soon as possible. Finally, a fluorescence microscope (ECCIPSE 50, Japan) was used to capture the images of the resulting cells.

until the value remained stable under 233 nm. Subsequently, 2 mL of glycol was added to the solution in order to cease the periodate oxidation process. Thereafter, 0.0108 M NaOH was used to titrate 2 mL of the periodate product for the sake of quantifying the production of formic acid. Smith degradation was then performed on the rest of the periodate product. After being dialyzed in ultrapure water for 72 h, the resulting solution was concentrated to 10 mL by rotary evaporation and then the excess furfural was reduced with the addition of 70 mg of NaBH4 and kept overnight. Then, the solution was neutralized to pH 6.5 by 50% acetic acid followed by being dialyzed against deionized water for another 72 h. Thereafter, the sample was totally dried by rotary evaporation and hydrolyzed by TFA (4 mL, 2 M) at 110 °C for 5 h. The resulting product was acetylated and detected by the gas chromatography (GC) (Agilent, USA) system, as mentioned above.

2.4.3. Investigation of pattern recognition receptor for TPS-2 Different antibodies (anti-SR, anti-MR, anti-GR, anti-TLR2, antiCR3, and anti-TLR4) at a concentration of 5 μg/mL, were added to the cells to bind with the corresponding membrane receptors, and were incubated for 2 h. Subsequently, the resulting macrophage cells were stimulated by TPS-2 (125 μg/mL). The group treated with only TPS-2 (125 μg/mL) was treated as the control. The untreated cells and LPStreated cells were used as the negative and positive control groups, respectively.

2.3.6. Methylation analysis The methylation was performed according to the method reported by Lin et al. (2012), with some modification. Ten milligrams of TPS-1 or TPS-2 was dissolved in 5 mL of DMSO using an ultrasonic water bath. Subsequently, 200 mg of NaOH was added to the solution, which was treated with an ultrasonic wave for 30 min. Thereafter, 2.5 mL of methyl iodide was added to this mixture and kept in darkness for 12 h at 25 °C. The resulting solution was extracted four times with chloroform, and the chloroform layer was washed four times with ultrapure water. All chloroform layers were collectively dried in a rotary evaporator. The dry product was hydrolyzed by TFA (4 M, 4 mL) at 100 °C for 2 h. In order to remove the excess TFA, the hydrolysate was evaporated to dryness, redissolved in methanol, and then dried out again. The residue was dissolved in 4 mL of deionized water, and the pH was adjusted to 10 using 10% NaOH. Thereafter, 100 mg of NaBH4 was mixed with the sample and agitated at a constant speed for 6 h. The pH of the solution was adjusted to 5.5 with 50% glacial acetic acid, and the solution was dried using a rotary evaporator. The dry product was thrice dissolved in methanol and then dried out. The resultant product was again dissolved in 2 mL of methanol and then dried out using nitrogen. The acetylization of the dry product was carried out by adding 2 mL acetic anhydride and 2 mL pyridine to it, and incubating at 90 °C for 30 min. The acetylization reaction was stopped by adding 2 mL of ultrapure water. The acetylated derivatives were extracted twice with 4 mL of methylene chloride. Finally, the resulting solution was analyzed with a GC–MS instrument (Agilent, USA) using a HP-5 capillary column (30 m × 320 μm × 0.25 μm), and the temperature programming was the same as mentioned above.

2.4.4. Data analysis Data were expressed as means ± standard deviation (SD) for three replicates. Data analysis was performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) through one-way analysis of variance (ANOVA) followed by the Duncan’s multiple range test. Unless otherwise stated, p-values < .05 were considered statistically significant. 3. Results and discussion 3.1. Preparation of TPS-1 and TPS-2 The crude polysaccharides were obtained from the dry corms of taro by hot water extraction followed by ethanol precipitation with a yield of 4.12% (w/w). The crude polysaccharides were first purified using a DEAE Sepharose Fast Flow column (Fig. 1A). The fractions TPS-1, TPS2, and TPS-3 were eluted using deionized water, 0.1 M NaCl, and 0.2 M NaCl, respectively. The fractions eluted with deionized water or 0.1 M NaCl showed a much higher content than the fractions eluted with 0.2 M NaCl; therefore, we mainly focused on the fractions TPS-1 and TPS-2 in the present study, and TPS-3 will be analyzed in a further study. The TPS-1 and TPS-2 fractions were further purified using a Sephacryl S-200 high resolution column. A single peak of TPS-1 or TPS2 was observed (Fig. 1B and C). After freeze-drying the TPS-1 and TPS-2 fractions, their polysaccharide contents were determined to be 94.3 and 95.4%, respectively. 3.2. Chemical structure of TPS-1 and TPS-2

2.3.7. NMR spectroscopy Each polysaccharide (30 mg) was dissolved with D2O (600 μL) in an NMR tube. The one-dimensional (1D) spectra were recorded on a Bruker 600 MHz NMR spectrometer (Bruker Corp, Switzerland).

3.2.1. Determination of purity and molecular weight A UV spectrophotometer was used to scan the solution of each polysaccharide in the range of 200–800 nm. According to Figs. S1 and S2, the UV absorption spectra of TPS-1 and TPS-2 lacked absorption peaks both in the wavelengths of 260 nm and 280 nm, indicating that TPS-1 and TPS-2 did not contain any interfering nucleotides or proteins. The weight-average molecular weights of TPS-1 and TPS-2 were 10.502 and 10.191 kDa, respectively, as determined by HPGPC (Fig. 2A and B). However, a previous study by Park et al. (2013) reported that the molecular weight of one of the taro polysaccharides was 200 kDa. Several reasons, such as the distinction of plant area, species, extraction, purification method, and so on, might account for this great difference. Furthermore, another previous study proved that geographical differentiation might lead to phylogenetic diversity among taro plants (Ochiai, Nguyen, Tahara, & Yoshino, 2001). Both TPS-1 and TPS-2 are low-molecular-weight polysaccharides; however, in most cases, botanical polysaccharides can reach a molecular weight of 100–1000 Da. This result hinted that TPS-1 and TPS-2 might present diverse characteristics from the high-molecular-weight polysaccharides. One previous study reported that some low molecular weight polysaccharide

2.4. Immunomodulatory activity of TPS-1 and TPS-2 2.4.1. Determination of NO and cytokines level RAW 264.7 cells were incubated in a culture medium (DMEM plus with 100 μg/mL of streptomycin, 10% fetal bovine serum, 100 units/mL penicillin) at 37 °C with 5% CO2. Cells were seeded in 96-well microplates or 6-well microplates at a density of 1 × 106 cells/mL in the exponential phase. After incubating the cells for 24 h, they were treated with step concentrations of TPS-1 or TPS-2 (62.5, 125, 250, 500, or 1000 μg/mL) and incubated for another 24 h. After the cells supernatants were collected, the Griess method and ELISA kit were used to determine the secretion levels of NO, TNF-α, and IL-6. The cells treated with LPS served as the positive control group. 2.4.2. Phagocytic capacity determination of TPS-2 Different concentrations of TPS-2 (62.5, 500, 1000 μg/mL) or LPS (20 μg/mL) were added to stimulate the cells, which were then 49

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except for the molecular masses, there were no other similarities to be found between the two polysaccharides and tarin. In fact, similar molecular-weight polysaccharides are also found in many other plants, like maca and orchid roots. The molecular masses of TPS-1 and TPS-2 are common in the polysaccharides of plants, and may be not related to tarin (Georgiadis et al., 2012; Zhang, Wang, Lai, & Wu, 2016; Zhang et al., 2017). 3.2.2. FT-IR spectrum analysis In the FT-IR spectrum of the TPS-1 and TPS-2 polysaccharides shown in Fig. 2C and D, most of the absorption peaks could be determined on the basis of previous reports. The IR spectrum of TPS-1 and TPS-2 displayed a broad stretching peak at 3406 and 3383 cm−1, attributed to the OeH stretching vibration of polysaccharides (Seedevi, Moovendhan, Vairamani, & Shanmugam, 2016). The absorption bands at 2926 and 2930 cm−1 correspond to the CeH stretching vibration in TPS-1 and TPS-2, respectively. The strong bands at 1645 and 1644 cm−1 might be attributed to the stretching vibration of C ] O or δ(HOH) (Wang, Chen, Wang, & Xing, 2015). The peaks at 932 and 763 cm−1 of TPS-1, and 932 and 761 cm−1 of TPS-2, were considered characteristics of the pyranose forms of glucosyl residues (Zhu, Xue, & Zhang, 2016). The absorption bands at 855 and 850 cm−1 were assigned to the existence of an α-configuration (Zhu et al., 2016). Taken together, the results of the FT-IR spectrum assay proved that both TPS-1 and TPS-2 contained the typical groups of sugars. 3.2.3. Monosaccharide composition The monosaccharide compositions of TPS-1 and TPS-2 were determined by gas chromatography (GC). According to Fig. 3, TPS-1 consisted of Rha, Xyl, Glc, and Gal. Four kinds of monosaccharides, Rha, Arf, Glc and Gal, were identified in TPS-2. The relative molar percentages of Rha, Glc, and Gal were 8.50%, 88.57%, and 2.56% in TPS-1 and 3.54%, 87.92%, and 6.94% in TPS-2, respectively. Moreover, the relative molar percentage of Xyl in TPS-1 was 0.73% and Arf in TPS2 was 1.60%. This result suggests that Glc constitutes the backbone structure of both TPS-1 and TPS-2. In addition, this result showed a significant difference from those of (Park et al., 2013), who identified that Taro-4-I mainly comprised of Gal (38.9%), Man (19.2%), Glc (4.2%), and uronic acid (35.6%). 3.2.4. Periodate oxidation-smith degradation analysis The periodate oxidation of TPS-1 showed that 1 mol of sugar residues consumed 0.3944 mol of periodate and produced 0.1637 mol of formic acid, whereas that of TPS-2 showed that 1 mol of sugar residues consumed 0.4188 mol of periodate and produced 0.1559 mol of formic acid. The production of formic acid indicated the presence of (1→) or (1 → 6) glycosidic linkages. The amount of periodate consumption was more than twice the production of formic acid, suggesting the existence of (1 → 2), (1 → 2, 6), (1 → 4), or (1 → 4, 6) glycosidic linkages in TPS-1 and TPS-2. The periodate-oxidized product of TPS-1 or TPS-2 was further analyzed by Smith degradation (Fig. 5D and E). The presence of glycerol indicated that (1 → 2) or (1 → 4)-linked glycosidic bonds might exist in both TPS-1 and TPS-2. The presence of Rha declared that it was linked by the manners of (1 → 3) in the two polysaccharides. However, more monosaccharides were detected from the product of the TPS-2 Smith degradation, suggesting that Gal (1 → 3) and Arf (1 → 3) might be the components of TPS-2. More accurate glycosidic linkages were identified through methylation analysis and NMR spectroscopy analysis.

Fig. 1. Chromatography of the polysaccharides from Colocasia esculenta by DEAE Sepharose Fast Flow (A); Sephacryl S-200 High Resolution chromatography of TPS-1 (B) and TPS-2 (C). Polysaccharide content was detected at the absorbance of 490 nm by using the phenol-sulfuric acid method.

fractions (< 20 kDa) are connected to higher immunological activities (Stephanie, Eric, Sophie, Christian, & Yu, 2010). Although the molecular weights of TPS-1 and TPS-2 were close to that of tarin subunits, a taro lectin that has immunomodulatory activity (Pereira et al., 2014; Pereira, Silva, et al., 2015; Pereira, Winter, et al., 2015), the results of the following structural analysis indicated that TPS-1 and TPS-2 did not have peptide bond structures. This meant that TPS-1 and TPS-2 did not contain lectin. Further, tarin only contains a 2–3% carbohydrate content, which mainly consists of mannose (Pereira, Winter, et al., 2015). The carbohydrate contents of TPS-1 and TPS-2 were higher than 94%, and did not contain mannose. Thus,

3.2.5. Methylation analysis Methylation analysis is an effective way to further confirm the location of glycosidic linkage in polysaccharides (Vieira, Mulloy, & Mourao, 1991). Table 1 shows the glycosidic bonds of TPS-1 and TPS-2, respectively, as determined by the GC–MS system. A significant difference was observed between the glycosidic linkages of TPS-1 and TPS50

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Fig. 2. HPGPC (A) of TPS-1; HPGPC (B) of TPS-2; FTIR spectrum of TPS-1 (C) and TPS-2 (D). A TSK G-5000PWXL column (7.8 × 300 mm), TSK G-3000PWXL column (7.8 × 300 mm) and Waters 2414 differential refractive index detector were applied to HPGPC. FT-IR spectrum was measured by a Fourier transform infrared spectrometer in 4000–400 cm−1 region.

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Fig. 3. Gas chromatograms of standard monosaccharides, phycite, glycerol and glycol (A); Gas chromatograms of TPS-1 (B) and TPS-2 (C) samples. The production of TPS-1 (D) and TPS-2 (E) after Smith degradation. In the measurement of the monosaccharide composition of TPS-1 and TPS-2, polysaccharides were hydrolyzed by trifluoroacetic acid, and the products were analyzed using gas chromatography equipped with an HP-5 capillary column and a flame ionization detector. In periodate oxidation-smith degradation analysis, TPS-1 or TPS-2 was oxidized by NaIO4. Thereafter, Smith degradation was performed on the periodate product. The degradation products were detected by gas chromatography.

2. Rha and Gal were detected as Rha-(1 → 3) and Gal-(1 → 2) in TPS-1, but as Rha-(1 → 2) and Gal-(1 → 3) in TPS-2. Moreover, Glc was identified as Glc-(1→), Glc-(1 → 6), and Glc-(1 → 2, 6) in TPS-1, but as Glc(1→), Glc-(1 → 4) and Glc-(1 → 4, 6) in TPS-2. Furthermore, the molar ratio of each linkage in TPS-1 was not in coincidence with TPS-2. The obvious differences in the structures of the two polysaccharide fractions indicated that TPS-1 and TPS-2 might have diverse biological activities.

signal at 5.12 ppm confirmed the presence of α-D-Glcp. The 13C NMR spectrum of TPS-1 is shown in Fig. 4B. The anomeric proton chemical shifts at 4.86 and 5.12 ppm, with a 13C chemical shift around 101.16 ppm, were attributed to (1→)-α-D-Glcp and (1 → 2, 6)-α-D-Glcp linkages, respectively. The anomeric proton peaks at 5.12 and 4.70 ppm followed by the anomeric carbon peaks at 97.5 and 100.17 ppm, corresponding with H-1 and C-1, revealed that TPS-1 contained (1 → 6)-αD-Glcp glycosidic bonds and (1 → 4)-β-D-Xylp, respectively. In addition, the 13C NMR spectrum signals appearing around 107.41 ppm with different anomeric H-1 signals (4.86 and 5.23 ppm) were attributed to the (1 → 3)-β-L-Rhaf and (1 → 2)-β-D-Galf, respectively, probably because the anomeric carbon peaks of these two types of glycosidic linkages were close to each other, and the Gal sugar residues were in low abundance. The 1H NMR and 13C NMR spectra of TPS-2 are presented in Fig. 4C and D. The H-1 (δ 5.14, 5.32, and 5.14) and C-1 signals (δ 98.68, 107.36, and 104.43) corresponded to the three anomeric residues, (1 → 2)-α-L-Rhaf, (1 → 3)-α-L-Araf, and (1 → 3)-α-Galf, respectively. The presence of carbon resonance at 104.43 ppm indicated that Glc residues existed in the β configuration in TPS-2. Based on this result, the presences of 4.54, 4.68, and 4.56 ppm were identified as (1→)-D-β-Glcp, (1 → 4)-D-β-Glcp, and (1 → 4, 6)-D-β-Glcp, respectively. The entire attributions of chemical shifts of TPS-1 and TPS-2 are summarized in Table 2.

3.2.6. NMR spectroscopy analysis More detailed structural information on TPS-1 and TPS-2 was derived based on the NMR spectra. The signals of 13C NMR and 1H NMR spectra were identified by comparing with data from existing literature (Dong et al., 2012; Gong et al., 2015; Jing et al., 2014; Svensson, Zhang, Huttunen, & Widmalm, 2011; Yuan et al., 2015; Zhang, Liu, & Lin, 2014). As shown in Fig. 4A–D, the spectra of TPS-1 and TPS-2 were different from each other. For TPS-1, according to its 1H NMR spectrum (Fig. 4A), the chemical shifts at 4.86, 4.70, and 5.23 ppm were referred to as anomeric H-1 of β-L-Raf, β-D-Xyl, and β-D-Galf, respectively. The

3.2.7. Identification of triple-helix conformation According to previous reports, the polysaccharides with triple-helical conformations could form a complex with Congo red and increase the maximum absorption wavelength in aqueous or weakly alkaline solutions, and decrease it in highly alkaline solutions (Liu et al., 2016; Wang et al., 2017). The shifts of the maximum absorption wavelengths of Congo red, Congo red + TPS-1, and Congo red + TPS-2 at various concentration of NaOH, ranging from 0 to 0.4 M, are shown in Fig. 4E. The maximum absorption wavelength of Congo red + TPS-1 and Congo red + TPS-2 decreased gradually with an increase in NaOH

Table 1 Glycosidic linkage composition of methylated TPS-1 and TPS-2. Sample

Residues

Methylated sugar

Linkage

Molar ratio

TPS-1

L-Rhap D-Xylf D-Glcp

2,4,6-Me3-Rhap 2,3-Me2-Xylf 2,3,4,6-Me4-Glcp 2,3,4-Me3-Glcp 3,4-Me2-Glcp 3,4,6-Me3-Galp

1,31,4T1,61,2,61,2-

8.62 1.31 9.24 60.58 17.99 2.27

3,4,6-Me3-Rhap 2,4-Me2-Rhap 2,3,4,6-Me4-Glcp 2,3,6-Me3-Glcp 2,3-Me2-Glcp 2,4,6-Me3-Galp

1,21,3T1,41,4,61,3-

3.67 2.13 12.25 54.98 19.54 7.44

D-Galf TPS-2

L-Rhap L-Araf D-Glcp

D-Galf

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Fig. 4. 1H NMR spectrum (A) and 13C NMR spectrum (B) of TPS-1; 1H NMR spectrum (C) and 13C NMR spectrum (D) of TPS-2, NMR Spectroscopy recorded on a Bruker 600 MHz NMR spectrometer; Maximum absorption wavelength of polysaccharide − Congo red complex at different concentrations of NaOH ranging from 0 to 0.4 M (E).

synthetic compounds, most botanical polysaccharides exhibit relatively non-cytotoxic effects and do not have any obvious side-effects (Schepetkin & Quinn, 2006). An assessment of polysaccharide toxicity can be regarded as an indicator of their biological and therapeutic properties (M. Wang et al., 2017). An MTT assay was performed to determine the viability of RAW 264.7 cells when treated with different concentrations of TPS-1 or TPS-2 (62.5, 125, 250, and 500, 1000 μg/ mL). As shown in Fig. 5A, both TPS-1 and TPS-2 did not induce mortality in RAW 264.7 cells at concentrations ranging from 62.5 to 1000 μg/mL, indicating that these two fractions of polysaccharides had nontoxic effects on the cells. In order to maintain cells in a normal growth state, the concentrations of TPS-1 and TPS-2 used in the subsequent experiments were kept under 1000 μg/mL.

concentration, and then reached close to the maximum absorption wavelength of Congo red. Therefore, both TPS-1 and TPS-2 have nontriple-helix conformations. Although previous studies indicated that triple-helix conformation may contribute to higher immunostimulation activity, it is suggested that monosaccharide composition may surpass the influence of helical conformations for the exhibition of immunostimulatory activity (Falch, Espevik, Ryan, & Stokke, 2000; Ferreira, Passos, Madureira, Vilanova, & Coimbra, 2016).

3.3. Immunomodulatory activity of TPS-1 and TPS-2 3.3.1. Cytotoxic effects of TPS-1 and TPS-2 on RAW 264.7 cells Compared with immunomodulatory bacterial polysaccharides and 53

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Table 2 Chemical shifts of resonances in the 1H and Sample

Sugar residue

13

C NMR spectra of TPS-1 and TPS-2. Chemical shift (ppm) C1/H1

C2/H2

C3/H3

C4/H4

C5/H5

C6/H6

TPS-1

→3)-β-L-Rha(1→ →4)-β-D-Xyl(1→ α-D-Glc(1→ →6)-α-D-Glc(1→ →2,6)-α-D-Glc(1→ →2)-β-D-Gal(1→

107.41/4.86 100.17/4.70 101.16/4.86 97.51/5.12 101.16/5.12 107.41/5.23

76.22/4.06 72.76/3.29 74.59/3.53 72.76/3.85 76.91/4.06 91.95/3.93

82.28/3.66 74.55/3.48 72.94/3.85 74.06/3.55 69.39/3.85 76.91/4.06

76.69/4.06 70.43/3.66 71.58/3.46 71.58/3.53 74.59/3.55 82.28/3.91

73.36/3.69 64.60/3.91 75.39/3.73 70.00/3.75 63.40/3.73 71.24/3.85

–/– –/– 63.40/3.75 60.80/3.93 76.22/3.93 64.60/3.91

TPS-2

→2)-α-L-Rha(1→ →3)-α-L-Arf(1→ β-D-Glc(1→ →4)-β-D-Glc(1→ →4,6)-β-D-Glc(1→ →3)-α-D-Gal(1→

98.68/5.14 107.36/5.32 104.43/4.54 104.43/4.68 104.43/4.56 99.71/5.14

81.41/3.70 78.82/4.56 74.18/3.32 74.18/3.35 74.18/3.33 70.47/3.51

68.42/4.09 83.34/3.94 77.11/3.51 76.86/3.56 76.86/3.56 80.26/4.09

70.47/3.89 82.40/4.56 70.47/3.32 80.26/3.89 80.26/3.89 69.65/3.86

69.64/3.86 60.53/3.89 75.83/3.62 75.80/3.76 76.45/3.70 75.80/4.09

–/– –/– 62.39/3.76 60.53/3.88 69.64/3.94 65.08/3.94

TPS-1 showed a significant difference compared to the control group, indicating that TPS-1 can induce macrophages to release NO and cytokines at concentrations ranging from 250 to 1000 μg/mL. However, the treatment with TPS-1 improved the production of cytokines NO, TNF-α, and IL-6 to a slight degree compared to treatment with TPS-2. The treatment with TPS-2 at a concentration of 62.5–1000 μg/mL resulted in an apparent induced-effect on the production of NO, TNF-α, and IL-6. The treatment with 62.5 μg/mL of TPS-2 led to increases in the production of NO, IL-6, and TNF-α of 0.52-, 0.14-, and 0.58- fold, respectively, compared to the LPS-stimulated group. Further, this increase in the NO generation and cytokine secretion properties of TPS-2 can be described as follows: at low concentrations of TPS-2, NO and cytokine productions increased rapidly, whereas at medium and high concentrations, this increase was slow. Based on the above-mentioned results, both TPS-1 and TPS-2 can modulate the immune response by activating macrophages to varying degrees. Especially, TPS-2 exhibits stronger immunocompetence than TPS-1. Our results were consistent with previous studies that reported that the plant-derived

3.3.2. TPS-1 and TPS-2 induced the up-regulation of NO and cytokines in RAW 264.7 cells A previous study reported that macrophages play a unique role in innate immunity and contribute to host defense (Cassetta et al., 2011). Polysaccharides can stimulate macrophages to secrete NO and cytokines to improve their ability to defend the body against microbial infections and tumor cells (Xie et al., 2008). In the present study, we determined the effects of TPS-1 and TPS-2 on the secretion of NO and relative cytokines (IL-6, TNF-α) in macrophages. As shown in Fig. 5B–D, both the fractions of polysaccharides were able to activate macrophages to secrete NO, TNF-α, and IL-6 in a dose-dependent manner. The control group cells were not subjected to TPS-1 or TPS-2 treatment and secreted a basal level of TNF-α, IL-6, and NO. For TPS-1, no significant differences were detected between the low-concentration group (62.5 μg/mL or 125 μg/mL) and the control group for IL-6 and TNF-α secretion, and the same result was obtained between the lowconcentration group (62.5 μg/mL) and the control group for NO production (p < .05). In contrast, the treatments with 250–1000 μg/mL of

Fig. 5. Effect of TPS-1 and TPS-2 on the viability of RAW 264.7 cells (A); Effects of TPS-1 and TPS-2 on secretion levels of NO (B), IL-6 (C), TNF-α (D) in RAW 264.7 cells after being treated for 24 h. The group without TPS-1 or TPS-2 was considered as the negative control, and LPS (50 μg/mL) was used as the positive control group. The group without TPS-1 and TPS2 was considered as the negative control, and LPS (20 μg/mL) was used as the positive control group. (E) Fluorescence microscopic images of RAW 264.7 cells phagocytosing FITC-labeled E. coli. induced by TPS-2 (10 × 20). The secretion levels of NO and cytokines were determined by Griess method and ELISA kits using a microplate reader. Fluorescence microscopic images were photographed by a fluorescence microscope.

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Fig. 6. Roles of CR3, GR, TLR4, TLR2, MR and SR on the NO (A), IL-6 (B) and TNF-α (C) secretion in RAW 264.7 cells stimulated by TPS-2. The macrophages cells were mixed with antibodies of the receptors for 2 h and then washed with PBS three times following inducing by TPS-2 (125 μg/mL). The values are presented as mean ± SD (n = 3). *, p < .05 and **, p < .01, versus the negative control group; Significant differences with the group treated with only TPS-2 (125 μg/mL) were designated as Δ, p < .05 and ΔΔ, p < .01. The secretion levels of NO and cytokines were determined by Griess method and ELISA kits using a microplate reader.

receptors. Furthermore, studies have shown that some plant-derived polysaccharides with different structures can recognize and bind to various membrane receptors, which might contribute to inducing immunomodulatory responses in macrophages (Rodrigues & Grenha, 2015; Schepetkin & Quinn, 2006). These kinds of binding proteins mainly consist of the toll-like receptors (TLR-2 and TLR-4), scavenger receptor I (SR), β-glucan receptor (GR), complement receptor 3 (CR3), and mannose receptor (MR). In order to identify the surface receptor of TPS-2 on RAW 264.7 cells in the present study, the roles of the abovementioned six PRRs were investigated. According to Fig. 6A–C, compared to the group treated with only TPS-2, a significant reduction in IL-6 and TNF-α was observed after the treatment of macrophages with anti-GR, anti-TLR4, and anti-SR. Furthermore, the treatment of cells with anti-GR or anti-TLR4 also lowered the expression of NO. However, the treatment with anti-SR did not decrease the secretion of NO. Instead, the group treated with anti-TLR2 lowered the expression level of NO compared to the group treated with only TPS-2. The existence of other potential membrane receptors might account for these results. No decrease in the levels of NO, TNF-α, or IL-6 was detected in the groups treated with anti-CR3 and anti-MR. In conclusion, the above results indicate that GR, TLR-4, TLR-2, and SR are the main kinds of PRRs of TPS-2 in RAW 264.7 cells. However, the six receptors investigated in this study are just the major receptors of polysaccharides on macrophages. It has been reported that some other polysaccharides receptors are distributed on the membrane of macrophages. Therefore, in our futures studies, more TPS-2 receptors will be confirmed and investigated. It is reported that polysaccharides containing arabinose, galactose, and rhamnose can be recognized in TLR-2 and TLR-4 (Zhang et al., 2011; Zhang et al., 2014). Therefore, the existence of these monosaccharides in TPS-2 may be connected to the resultant immune response. In addition, previous studies have proven that polysaccharides containing glucose are recognized in TLR-2, TLR-4, GR, and SR (Brown, 2006; Chen et al., 2017; Rice et al., 2002). Thus, compared to other monosaccharides, the high content of glucose in TPS-2 may play a more decisive role in recognizing its PRRs on macrophages.

polysaccharides are able to promote immunological activity. The immunostimulatory activity of polysaccharides is tightly associated with their structural features, such as molecular weight, monosaccharide composition, glycosidic-linkage, conformation, branching degree, etc. (Ferreira et al., 2016). Interestingly, both TPS-1 and TPS-2 are low-molecular-weight polysaccharides with non-triple helix conformation and similar branching characteristics. Based on the results obtained, it was presumed that the difference in monosaccharide composition and glycosidic-linkage contribute to the distinct immunological activity between TPS-1 and TPS-2. Previous studies have reported that arabinose-containing polysaccharides exhibit an immuneenhancing effect (Ebringerova, Kardosova, Hromadkova, Malovikova, & Hribalova, 2002; Zhou et al., 2010). From this prospective, the presence of arabinose in TPS-2 and its absence in TPS-1 might indicate that TPS-2 was more effective in stimulating macrophages to secret NO as well as cytokines. One previous study reported that polysaccharides that possess (1 → 4)-β-D-Glc-branched (1 → 6)-β-D-Glc residues achieve greater results in performing immunological functions (Zhao, Chen, Ren, Han, & Cheng, 2010). Therefore, the possession of these two kinds of glycosidic bonds in TPS-2 might contribute to its satisfactory immunological activity. More detailed information on the different immunological activities exhibited by TPS-1 and TPS-2 will be explored in our future study. 3.3.3. Effects of TPS-2 on the phagocytic capacity of RAW 264.7 cells According to the above results, TPS-2 displays a strong immunological activity by promoting the secretion of NO, IL-6, and TNFα. However, the ability to internalize foreign substances is also essential to defend cells against pathogen infection (Yu et al., 2015). Therefore, the effect of TPS-2 on the ability of macrophages to phagocytize E. coli was investigated to further explore its immunological activity. As shown in Fig. 5E, the images of green fluorescence in RAW 264.7 cells were captured using a fluorescence microscope to obtain a visual evidence of phagocytosis; the denser the green fluorescence distribution, the stronger the ability of macrophages to phagocytize E. coli. Untreated RAW 264.7 cells (control) could phagocytose only a small amount of E. coli, whereas the cells treated with 62.5 μg/mL of TPS-2 showed effective phagocytosis. In addition, the treatment with 500 and 1000 μg/ mL of TPS-2 significantly enhanced the phagocytic activity of cells in a dose-dependent manner. These results indicated that TPS-2 has the ability to enhance the phagocytic activity of macrophages.

4. Conclusion In this study, two new polysaccharides, TPS-1 and TPS-2, were sequentially purified from the corms of taro. TPS-1 and TPS-2 were both non-starch polysaccharides and their molecular weights were estimated to be 10.502 and 10.191 kDa, respectively. TPS-1 consisted of Rha (8.5%), Xyl (0.73%), Glc (88.57%), and Gal (2.56%), while TPS-2 consisted of Rha (3.54%), Arf (1.6%), Glc (87.92%), and Gal (6.94%). The main glucosidic bonds of TPS-1 and TPS-2 were identified to be α(1, 6)-D-Glc and β-(1, 4)-D-Glc, respectively. TPS-2 exhibited a more effective immunomodulation activity than TPS-1 in terms of enhancing

3.3.4. Pattern recognition receptor for TPS-2 binding to macrophages Pattern recognition receptors (PRRs) are non-clonal immune proteins that participate in recognizing conserved the molecular structures (pathogen-associated molecular patterns) shared by a huge number of microorganisms (Leung, Liu, Koon, & Fung, 2006). Macrophages can be activated by the binding of botanical polysaccharides with specific 55

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NO, TNF-α, and IL-6 levels in RAW 264.7 cells. In addition, TPS-2 also showed a significant phagocytosis-stimulating ability for macrophages. TLR2, TLR4, GR and SR were determined to be the major membrane receptors of TPS-2. Based on their nontoxic effects on macrophages, TPS-1 and TPS-2 could be considered as novel potential immunostimulants as food and pharmaceutical supplements for hypoimmunity.

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