Structural characterization and immunoregulatory activity of a novel heteropolysaccharide from bergamot (Citrus medica L. var. sarcodactylis) by alkali extraction

Industrial Crops & Products 140 (2019) 111617

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Structural characterization and immunoregulatory activity of a novel heteropolysaccharide from bergamot (Citrus medica L. var. sarcodactylis) by alkali extraction

T

Bao Penga,1, Jianing Yangb,1, Weijuan Huangc,1, Dan Pengb, Sixue Bia, Liyan Songb, , Yao Wenc, ⁎⁎ ⁎ Jianhua Zhuc, , Yiyu Chena, Rongmin Yua,c, ⁎⁎

a b c

Biotechnological Institute of Chinese Materia Medica, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China Department of Pharmacology, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China Department of Natural Medicinal Chemistry, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China

ARTICLE INFO

ABSTRACT

Keywords: Citrus medica L. var. sarcodactylis Polysaccharide Structure characterization Immunoregulatory activity Food/pharmaceutical industries

Bergamot is an ornamental plant and important traditional Chinese herbal medicine of tremendous value in economic and medicinal applications, mainly distributed in southern and southeastern China. Polysaccharide is one of active components of bergamot. In this study, CMSPB80 was extracted from bergamot by alkali solution and purified by DEAE Sepharose fast flow and Sephadex G-75 column to obtain a homogeneous heteropolysaccharide (CMSPB80-1), with a molecular weight of 103 kDa. Ion chromatography analysis indicated that CMSPB80-1 was composed of →3)-α-L-Rhap-(1→, →5)-α-L-Araf-(1→, →3,6)-α-D-Manp-(1→, →3)-β-D-Galp-(1→ , →4,6)-α-D-Glcp-(1→, →6)-β-D-Galp-(1→ and a terminal α-D-Glcp. Scanning electron microscope (SEM) demonstrated that CMSPB80-1 was rough with a sheet-like and irregular structure. Atomic force microscopy (AFM) displayed CMSPB80-1 was branched and entangled with globular aggregates instead of a triple-helix structure. In addition, in vitro activity of CMSPB80-1 was evaluated by neutral red up-take assay and production of nitric oxide (NO) assay, which provided evidence that CMSPB80-1 could enhance macrophage phagocytiosis and promote splenocyte lymphocytes proliferation. The results proposed that CMSPB80-1 was suitable for health care and food/pharmaceutical industries as an immunoregulatory activity ingredient.

1. Introduction Polysaccharides are diffusely present in plants and microorganisms. They are formed by polymerization of more than 10 monosaccharides through glycosidic bond (Zong et al., 2012). The botanical polysaccharides are a class of natural macromolecular substances that are widely present in plant. In recent decades, reports on plant polysaccharides has attracted widespread attention and interest duo to their low toxicity and therapeutic properties (Schepetkin and Quinn, 2006). Plant polysaccharides have many biological activities, such as anti-inflammatory, anti-tumor, anti-oxidation, antibacterial, hypoglycemic, anti-osteoporosis, immune regulation and immune stimulation (Zhang et al., 2007; Thambiraj et al., 2015; Yang et al., 2015; Wu et al., 2005; Huang et al., 2018a,b). Thus, plant polysaccharides were called biological response modifiers (BRMs), which augment immune response

(Leung et al., 2006). The immune-enhancing activity is the most important action of polysaccharide and has become a hot issue of polysaccharide investigation (Abula et al., 2011). In particularly, plant polysaccharides could improve host defense against pathogens and modulate adaptive immunity (Shen et al., 2017). Moreover, due to few side-effect, more interest of use in botanical polysaccharides for immune-enhancing agents and functional foods for prevention and treatment of many human diseases such as cancer (Thambiraj et al., 2015). In addition, studies have indicated that polysaccharides can be combined with chemotherapy drugs to treat cancer, which can significantly reduce the side-effect caused by chemotherapy drugs, thereby achieving the purpose of increasing efficiency and reducing toxicity (Xie et al., 2017). Macrophages are phylogenetically conserved cells in multicellular organisms, which belong to the first line of host defense of neutrophils

Corresponding author at: Biotechnological Institute of Chinese Materia Medica, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China. Corresponding authors. E-mail addresses: [email protected] (L. Song), [email protected] (J. Zhu), [email protected] (R. Yu). 1 These authors contribute equally to this work. ⁎

⁎⁎

https://doi.org/10.1016/j.indcrop.2019.111617 Received 6 April 2019; Received in revised form 23 June 2019; Accepted 27 July 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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(Bi et al., 2018). In addition, they participated in both specific and nonspecific immune reactions (Chen et al., 2012). Activated macrophages play an important role in homeostasis maintenance, inflammation regulation and repair, which can clear pathogen or damaged cells clearance (Murray et al., 2014). Many researches suggested that plant polysaccharides exert immune enhancement effect by increasing production of NO, cytokines and chemokines in macrophages (Park, 2014; Zeng et al., 2014). In particularly, NO is an important signaling molecule of neural, vascular, and immune systems in various biological activities. When macrophages are activated, a large amount of NO is released, which could exert cytostatic effects such as kill microorganisms, tumor cells and also induce inflammation against external injuries (Chen et al., 1999). Citrus medica L. var. sarcodactylis Swingle (bergamot), known as “Fo-Shou” in Chinese, is mainly distributed in Guangdong, Fujian, Sichuan, and Zhejiang provinces, China. It is regarded as an ornamental plant because of its unique appearance and charming aroma. Due to its enormous economic and food/medicinal value, bergamot has been extensively researched. The theory of Traditional Chinese medicine (TCM) displays that bergamot has many active substances, such as flavonoids, hesperidin, polysaccharides, alkaloids and coumarins. Bergamot is traditionally treated conditions such as hypertension, tracheitis, asthma, and respiratory tract infections (He et al., 2014). Also, bergamot could strengthen liver, pancreatic, and stomach (Wu et al., 2013). We have previously obtained a polysaccharide from the hot water extract of C. medica L. var. sarcodactylis (CMSPW90-1) and modified it by sulfation. The results of this study demonstrated that its sulfated derivative (CMSPW90-M1) could effectively promote the proliferation of spleen cells (Peng et al., 2018). To expand the research of bergamot, we extracted crude polysaccharides from the residues of C. medica L. var. sarcodactylis (CMSPB) using alkali solution. CMSPB were purified via DEAE sepharose fast flow and Sephadex G-75 chromatography, and a novel heteropolysaccharide was obtained, named CMSPB80-1. Structure of the CMSPB80-1 was carried out via ion chromatography, methylation analyses and NMR (1H, 13C, HSQC, and HMBC). The microstructures of CMSPB80-1 were preliminarily clarified using scanning electron microscope (SEM) and atomic force microscope (AFM). In addition, the proliferation and phagocytosis of CMSPB80-1 were also evaluated.

neutralized with 5 mol/L of HCl solution and then concentrated. Ethanol was added at a final concentration of 80% and incubated at 4 °C for 48 h. The precipitation was deproteinized with Sevag reagent (chloroform/n-butanol at a ratio of 4:1, v/v). Finally, the crude polysaccharide (CMSPB80) was obtained after dialyzation and lyophilization. 2.3. Separation and purification of CMSPB80 CMSPB80 (10 g) was purified on a DEAE sepharose fast flow column (2.5 × 40 cm), eluted in succession with distilled water and a linear gradient from 0 to 0.8 M NaCl at a flow rate of 1.6 mL/min. The phenolsulfuric acid method detected the content of polysaccharides in the eluents (Yi et al., 2015). A sharp peak (CMSPB80-S) was lyophilized and further purified on a Sephadex G-75 permeation chromatography column (1.5 × 100 cm) eluted with distilled water at a flow rate of 0.3 mL/min. Consequently, a purified polysaccharide CMSPB80-1 was obtained after the concentration and lyophilization. 2.4. Determination of homogeneity and average molecular weight of CMSPB80-1 A UV-2401 spectrophotometer was used to detect the presence of nucleic acids and proteins of CMSPB80-1 (100 μg/mL). CMSPB80-1 (0.5 mg) was dissolved completely in 5 ml of distilled water, which was UV-scanned in the wavelength range of 200–350 nm. The homogeneity and molecular weight of CMSPB80-1 were determined by high-performance gel-permeation chromatography (HPGPC) performed on a Waters HPLC system outfitted with TSK G-5000PWxl column (7.8 × 300 mm) and TSK G-3000PWxl analytical columns (7.8 × 300 mm) in series. CMSPB80-1 (4 mg) was dissolved completely in 2 mL of KH2PO4 buffer solution (0.02 M). The injection volume is 10 μL, the column temperature is 35 ℃, and the flow rate is 0.4 mL/min. The relative molecular weight of CMSPB80-1 was calculated from the standard curve, which was set with dextran. 2.5. FT-IR analysis of CMSPB80-1 CMSPB80-1 was analyzed and recorded via a Perkin Elmer FT-IR Spectrometer in the wavelength range of 4000-400 cm−1 after dried under the infrared light and pressed with KBr powder (Hua et al., 2014).

2. Materials and methods 2.1. Plant materials and chemicals

2.6. Chemical analysis of CMSPB80-1

Bergamot was purchased from the TongRenTang medicinal store (Guangzhou, China). The material was identified by Professor R.M. Yu, College of Pharmacy, Jinan University (Guangzhou, China). DEAE Sepharose fast flow and Sephadex G-75 were purchased from Whatman Ltd. RAW 264.7 cell line was obtained from ATCC (Shanghai, China). Fetal bovine serum (FBS) and DMEM culture media were purchased from Gibco BRL Co. (USA). Concanavalin-A (ConA) and lipopolysaccharide (LPS) were obtained from Sigma Chemical Co. (USA). NO detection kit and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Beyotime Biotechnology Research Institute (Shanghai, China). Kunming mice (20.0 ± 2.0 g) were purchased from Medical Laboratory Animal Center of Guangdong (Guangzhou, China). All other chemicals and reagents used in the experiments were of analytical grade.

Trifluoroacetic acid (TFA, 2 M, 10 mL) was used to hydrolyze 5 mg of CMSPB80-1 at 120 °C for 6 h and excess TFA was removed with methanol by evaporating. The hydrolysate (20 μL) was injected on the Dionex ICS-2500 system and eluted with water and 200 mM NaOH (92:8, v/v) for HPAEC-PAD analysis after re-dissolved and filtered with a 0.45 μm filter (Yu et al., 2007; Yuan et al., 2010). To analyze the glycosidic linkages and absolute configuration of monosaccharide, CMSPB80-1 was methylated according to established methods and then detected by FT-IR (Bao et al., 2009; Wang et al., 2017). Disappearance of the hydroxyl stretching vibration absorptive band (3200-3700 cm−1) indicated successive methylation. The methylated products were hydrolyzed, reduced, acetylated and analyzed via GCeMS.

2.2. Preparation of crude polysaccharide CMSPB80

2.7. Nuclear magnetic resonance (NMR) spectroscopy

The powder of dried bergamot (8 kg) was treated with 95% ethanol (1:8, w/v) for 3 h to remove alcohol-soluble chemicals and the residues were extracted with hot water (1:15, w/v) for 4 h. The residues of bergamot were treated with 0.3 mol/L of NaOH solution overnight, repeated twice, centrifuged and pooled. The supernatant was

The dried CMSPB80-1 (50 mg) was dissolved in 0.6 mL of D2O and analyzed on a Bruker AVANCE 600 MHz NMR spectrometer (Bremen, Germany) to perform 1H NMR, 13C NMR, HSQC and HMBC, respectively. 2

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plates at a density of 1 × 105 cells/mL and incubated for 24 h. Cells were treated with different concentrations of CMSPB80-1 (0–500 μg/ mL) for 24 h. The medium and LPS (1 μg/mL) were used as blank and positive control, respectively. Then, 0.1% neutral red solution (100 μL/ well) was added and incubated at 37 °C for 30 min. And then, the neutral red reagent was discarded and cells were washed with PBS for three times. Cell lysis buffer (1% glacial acetic acid: ethanol = 1:1, 200 μL/well) was added and incubated at 37 °C for 1 h. The absorbance was measured at 540 nm. The phagocytic index was calculated with the equation: Phagocytic index (PI)% = A1/A0×100% where A1 is the absorbance of sample, A0 is the absorbance of blank control.

2.8. Congo red test Congo red test could determine the conformational transitions (Liu et al., 2016). CMSPB80-1 (0.5 mg/mL) were mixed with 1 mL, 50 μmol/ mL Congo red and 1 M NaOH to make the finally concentration of NaOH to 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mol/L, which was detected by a UV-2401 spectrophotometer in the region of 400–600 nm. 2.9. Conformational and morphological analysis of CMSPB80-1 The dried powder of CMSPB80-1 was placed on a metal stub and sputtered with gold powder for observing the micro-morphological characteristics by a scanning electron microscope (Philips XL-30) at 5.0 KV under high vacuum conditions (Chen et al., 2013). CMSPB80-1 solution (1 μg/mL) was stirred with a magnetic stirrer apparatus for 4 h and took 5 μL out to dry on a freshly cleaved mica substrate. AFM (BioScope Catylyst Bruker, Bil-lerica, MA) was determined for the ultrastructural images of CMSPB80-1 (Wang et al., 2012).

2.13. Determination of NO The NO concentration in the culture medium was measured by Griess reaction as previously described. Briefly, 100 μL supernatants and NO detection kit were mixed. Then, the absorbance was measured at 540 nm. The values were calculated according to the calibration curve with NaNO2.

2.10. Antioxidant activity of CMSPB80-1 in vitro 2.10.1. Measurement of DPPH• radical-scavenging activity CMSPB80-1 solution (20 μL, 0–3.2 mg/mL) was mixed with 180 μL of DPPH of ethanol solution (0.2 mM) into the 96-well plate to detect the scavenging activity of DPPH• free radical (Tu et al., 2016). The final concentrations in the wells of the sample were 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL. The solution was maintained in the dark for 0.5 h in the constant temperature shaker and measured at 517 nm in the spectrophotometer. The Vc group was the positive control with the same final concentrations. The DPPH• free radical scavenging capability was calculated by the following equation: Scavenging ability (%) = (A0-A1)/A0 × 100% where A0 is the absorbance in the absence of the test sample and A1 is the absorbance in the presence of the test sample.

2.14. Proliferation of splenocyte lymphocytes The proliferation of splenocyte lymphocytes was determined by the MTT assay. Kunming mice were sacrificed, and the spleens were acquired aseptically, then gently pressed with syring core and passed through a 200-mesh steel sieve to obtain single-cell suspensions. The erythrocytes were removed by red blood cell lysis buffer, then splenocyte was washed three times with PBS and centrifuged at 1500 rpm for 5 min. Finally, splenocytes were suspended to 5 × 106/mL with DMEM medium supplemented with 10% FBS. The cells were seeded in 96-well plates with the final concentration of CMSPB80-1 (0–500 μg/mL). Followed the cells were incubated at 37 °C with 5% CO2 for 48 h. Then, 20 μL MTT solution (5 mg/mL) was added into each well and continuously incubated at 37 °C for 4 h. After incubation, 96-well plates were centrifuged at 3000 rpm for 5 min. Then, the supernatant was removed and 200 u L of DMSO was added to every well, and the absorbance was measured with microplate reader at 570 nm. All experiments were repeated in triplicate.

2.10.2. Measurement of ABTS+• radical-scavenging activity According to the method of ABTS+• free radical scavenging activity (Li et al., 2012), 0.2 mL of ABTS (7.4 mmol/L) was added into 0.2 mL of K2S2O8 (2.6 mmol/L) and kept in the dark at room temperature for 12 h to obtain the ABTS+• reagent. Then the absolute ethanol was added to dilute 30 times until the absorbance of the mixture reached 0.7 ± 0.02 at 734 nm. ABTS+• reagent (160 μL) was respectively added to 40 μL of different concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL) of CMSPB80-1 solutions into the 96-well plate and shaken for 15 min to be detected at 735 nm. The Vc group was the positive control with the same final concentrations. ABTS+• free radical scavenging capability was computed by the following formula: Scavenging ability (%) = (A0-A)/A0×100% where A0 is the absorbance in the absence of the test sample and A is the absorbance in the presence of the test sample.

2.15. Statistical analysis All results are presented as the mean ± standard error of mean (SEM), and data were analyzed using one-way analysis of variance or Student’s T tests. P values less than 0.05 were considered statistically significant. 3. Results and discussion

2.11. Cell viability assay

3.1. Isolation, purification, homogeneity, and molecular weight of CMSPB80-1

Cell viability was determined by MTT assay. Briefly, RAW 264.7 cells were seeded into 96-well plates at a density of 2 × 104 cells/mL and incubated for 24 h. Cells were treated with different concentrations of CMSPB80-1 (0–500 μg/mL) for 48 h. Then, 20 μL MTT solution (5 mg/mL) was added into each well and incubated at 37 °C for 4 h. After adding 200 μL DMSO to each well, the absorbance at 570 nm was measured using a microplate reader.

The yield of the CMSPB80 obtained from the dried bergamot was 0.88% (w/w). After separation through a DEAE Sepharose fast flow column with 0-0.8 M NaCl gradient (Fig. 1A), CMSPB80-S was dialyzed and purified using a Sephadex G-75 column to obtain CMSPB80-1 (Fig. 1B). A single and symmetrical narrow peak was exhibited in the HPGPC profiles (Fig. 1C), implying CMSPB80-1 was a homogeneous polysaccharide. The average molecular weight was calculated as 103 kDa. In addition, no nucleic acids and protein were found in CMSPB80-1 for the reason of the absence of the absorption peak at 260 nm and 280 nm in the UV spectroscopy (Fig. 1D) (Han et al., 2016).

2.12. Phagocytic activity The phagocytic ability of RAW 264.7 cells was determined by neutral red up-take assay. RAW 264.7 cells were seeded into 96-well 3

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Fig. 1. (continued) Fig. 1. Chromatographies, UV and FTIR spectra of CMSPB80/CMSPB80-1. A: Chromatography of CMSPB80 from bergamot by DEAE sepharose fast flow chromatography; B: Chromatography of CMSPB80-1 by sephadex G-75; C: HPGPC of CMSPB80-1; D: UV spectrum of CMSPB80-1; E: HPAEC of standard monosaccharides and CMSPB80-1 peaks: (1) Fucose, (2) Rhamnose, (3) Arabinose, (4) Galactose, (5) Glucose, (6) Mannose, (7) Fructose; F: FTIR spectrum of CMSPB80-1.

3.2. Chemical composition analysis of CMSPB80-1 The monosaccharide composition of rhamnose, arabinose, galactose, glucose, and mannose in CMSPB80-1 by HPAEC-PAD were at the molar ratio of 1.68:21.91:20.54:2.36:1.00 (Fig. 1E), indicating that CMSPB80-1 was a heteropolysaccharide.

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Table 1 Methylation analysis of CMSPB80-1. Methylation sugar

Retention time(min)

Molar ratio

Mass fragments(m/z)

Linkage type

2,3,4,6-Me4-Glc 2,4-Me2-Rha 2,3-Me2-Ara 2,4-Me2-Man 2,4,6-Me3-Gal 2,3-Me2-Glc 2,3,4-Me3-Gal

23.95 26.45 28.62 31.89 34.17 31.45 38.20

2.02 1.59 21.62 1.00 12.06 1.14 8.14

57,71,87,101,117,129,161 43,75,85,117,131,189 87,101,117,129,189 57,71,87,99,117,129,159 57,71,87,101,117,129,189 58,71,85,99,117,127,159 57,71,85,101,117,131,161

T→ 1→3 1→5 1→3,6 1→3 1→4,6 1→6

Fig. 2. 1H NMR (A) and

13

C NMR (B) of CMSPB80-1. Fig. 3. HSQC (A) and HMBC spectra (B) of CMSPB80-1.

3.3. FT-IR spectrum analysis of CMSPB80-1

3.4. Methylation and GC–MS analysis of CMSPB80-1

The FT-IR spectrum of CMSPB80-1 (Fig. 1F) showed absorption bands at 3422.03, 2928.20, 1632.53 and 1044.31 cm−1, which were characteristic absorption peaks typical of polysaccharides (Ji et al., 2018). The major broad stretching peak at 3422.03 cm−1 was the stretching vibration of OeH. The band at 2928.20 cm−1 was attributed to the stretching vibrations of CeH, and the band at 1632.53 cm−1 was due to the deformation vibration of OeH. There was no absorption at 1740 cm−1 indicating the absence of uronic acid in the polysaccharide structure. The absorptions at 1044.31 cm−1 suggested CeO bending vibrations (Jing et al., 2014). The typical absorption peak at 856.29 cm−1 and 893.60 cm−1 indicated that α- and β-configurations present simultaneously in CMSPB80-1 (Zhang et al., 2016).

More structural information of CMSPB80-1 on the percentage of glycosidic linkages was obtained from methylation analysis by GC–MS. Seven methylated alditol acetates were detected, including tri-O-acetyl2,3,4,6-tetra-O-methyl-D-glucitol, 1,3,5-tri-O-acetyl-2,4-di- O-methyl-6deoxy-L-mannitol, 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L-arabinitol, 1,3,6tri-O- acetyl-2,4-di-O-methyl-6-deoxy-L-mannitol, 1,3-di-O-acetyl-2,4,6tri-O-methyl-D-galactitol, tri-O-acetyl-2,3-di-O-methyl-D-glucitol, 1,6-diO-acetyl-2,3,4-tri-O-methyl-D-galactitol. The results revealed that CMSPB80-1 may be formed of D-Glcp-(1 →, →3)-L-Rhap-(1→, →5)- LAraf-(1→, →3,6)-D-Manp-(1→, →3)-D-Galp-(1→, →4,6)-D-Glcp-(1→ 5

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Table 2 1 H and 13C NMR chemical shifts of CMSPB80-1 (ppm). Sugar residue

→5)-α-L-Araf-(1→A →3)-β-D-Galp-(1→B →6)-α-D-Galp-(1→C →4,6)-β-D-Glcp-(1→D →3)-α-L-Rhap-(1→E →3,6)-β-D-Manp-(1→ F α-D-Glcp-(1→ G

Chemical Shifts C1/H1

C2/H2

C3/H3

C4/H4

C5/H5

C6/H6

109.26/5.16 107.42/5.01 106.97/5.09 106.38/5.16 104.19/4.55 103.30/4.40

81.42/4.12 76.57/4.09 76.19/3.94 76.34/4.09 77.67/3.63 69.37/3.68

84.91/4.08 83.93/3.80 80.96/4.12 84.91/4.05 84.01/4.23 82.26/3.99

72.46/3.59 76.49/3.94 76.68/4.09 69.71/3.58 76.68/3.72 76.46/3.88

80.17/4.05 80.94/4.05 83.93/4.17 80.17/4.14 61.03/4.00 71.02/3.68

68.31/4.04 65.99/3.72 76.68/4.09 16.49/1.17 71.83/3.45

95.50/5.13

83.87/3.88

71.02/3.68

82.26/3.88

66.79/3.81

61.13/3.75

Fig. 4. The predicted structure of CMSPB80-1.

HSQC spectrum was assigned to the C-1 of α-D-Glc, and the (1→) linked α-D-Glc was confirmed via the HMBC spectrum and GCeMS analysis (Huang et al., 2018a,b). In addition, the weak correlation peak at 1.17/ 16.49 ppm was easily attributed to the methyl groups of α-L-Rhap (H6/ C6). It was further assigned to the (1→3) linked α-L-Rhap based on the data from HSQC and HMBC spectra, which was consistent with methylation and GCeMS results. Accordingly, complete descriptions of residues A, B, C, D, E, F, and G are showed in Table 2, based on the methylation analysis data, HSQC, HMBC spectra and results reported in the literature (Li et al., 2017a,b; Zhu et al., 2017; Bi et al., 2013; Kang et al., 2011; Mandal et al., 2012). Finally, the HMBC spectrum of CMSPB80-1 was analyzed for the linkage sequence among the sugar residues. The strong signal at 5.16/ 80.17 ppm (A H1/A C5) showed that C-5 of residue A was linked to O-1 of residue A. Meanwhile, the methylation and GC-MS results revealed that (1→5) linked arabinose accounted for 45.45% of the total sugar residues. Thus, the presence of a repetitive unit of (1→5) linked arabinose was confirmed. Similarly, The strong signal at 5.01/83.93 ppm (B H1/B C3) showed that C-3 of residue B was linked to O-1 of residue B. The strong signal at 5.09/65.99 ppm (C H1/C C6) showed that C-6 of residue C was linked to O-1 of residue C, and the strong correlation peak at 3.72/106.97 ppm (C H6/C C1) suggests C-1 of residue C was linked to O-6 of residue C. Thus, the presence of a repetitive unit of (1→ 3) linked galactose and (1→6) linked galactose were also confirmed. Accordingly, the cross peaks at 5.16/65.99 ppm (A H1/C C6), 5.09/ 82.26 ppm (C H1/F C3), 3.58/103.30 ppm (D H4/F C1), 3.80/ 106.38 ppm (B H3/D C1), 4.05/107.42 ppm (A H5/B C1), 3.45/ 104.19 ppm (F H6/E C1), 5.13/84.01 ppm (G H1/E C3) and 4.55/ 76.68 ppm (E H1/D C6) suggest that C-6 of residue C was linked to O-1 of residue A; C-3 of residue F was linked to O-1 of residue C; C-1 of residue F was linked to O-4 of residue D; C-1 of residue D was linked to O-3 of residue B; C-1 of residue B was linked to O-5 of residue A; C-1 of residue E was linked to O-6 of residue F; C-3 of residue E was linked to O-1 of residue G and C-6 of residue D was linked to O-1 of residue E. Finally, the structure of CMSPB80-1 was conferred with the combination of molecular weight, monosaccharide composition, FTIR spectroscopy, methylation and NMR data, as shown in Fig. 4.

Fig. 5. Changes in absorption wavelength maximum of mixtures of Congo red and CMSPB80-1 at various concentrations of NaOH.

and →6)-D- Galp-(1→ at a ratio of 2.02:1.59:21.62:1.00:12.06:1.14:8.14 (Table 1). 3.5. NMR spectroscopy analysis HSQC and HMBC NMR spectra were used to determine the structural characterization of CMSPB80-1. As shown in 1H NMR spectra (Fig. 2A), the overlap of most proton signals lead to confirm the anomeric signals of CMSPB80-1 difficultly. In addition, some signals of anomeric carbon in 13C NMR spectra were too weak to be distinguishable (Fig. 2B). As shown in Fig. 3A, seven correlation peaks were observed at 5.16/109.26 ppm, 5.01/107.42 ppm, 5.09/106.97 ppm, 5.16/106.38 ppm, 4.55/104.19 ppm, 4.40/103.30 ppm and 5.13/95.50 (coded as A, B, C, D, E, F, G, respectively). The result revealed that CMSPB80-1 had seven sugar residues, corresponding with the results of methylation and GCeMS analysis. The signal at 5.13/95.50 ppm in the 6

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Fig. 6. The microscopic structure of CMSPB80-1 by SEM images (A: 300×, B: 500×); The microscopic molecular morphology of CMSPB80-1 by AFM (C, D: Scan size 5 μm).

3.6. Conformational analysis The spatial stereochemistry of the polysaccharide was determined by employing CR-polysaccharide complexes spectrophotometric method at various alkali concentrations (Wang et al., 2016). As shown in Fig. 5, the maximum absorption wavelengths of CMSPB80-1 complexes red-shifted to longer wavelengths compared with pure Congo red. However, the red shifts of λmax of the samples were not observed at all concentrations. Therefore, it could be concluded that no triple-helical conformation exists in the solution of CMSPB80-1 3.7. Morphological analysis The SEM images of the CMSPB80-1 at magnifications of 300- and 500-fold are shown in Fig. 6A and 6B. SEM images revealed that CMSPB80-1 was mainly composed of many small lumpish and irregularly distributed flaky particles, implying an amorphous structure in CMSPB80-1 (Huang et al., 2018a,b). Atomic force microscopy (AFM) is developed on the basis of scanning tunneling microscope, and it is used as a novel material structure tool to analyze the structures of polysaccharides and function of biological macromolecules (Wang et al., 2018a,b). Fig. 6C showed the molecular morphology (scanned at 5 × 5 μm) of CMSPB80-1. There were some dispersions and spherical lumps. Fig. 6D showed a tridimensional structure of CMSPB80-1, which represented the small amount of globular aggregates with diameters ranging from 200 to 300 nm and height ranging from 12.0 to 22.0 nm in the solution. The surface topography was rugged and irregular. Spherical and uneven lumps also suggested that molecular aggregation and the branches had occurred in the chemical structures of CMSPB80-1 (Li et al., 2017a,b).

Fig. 7. Antioxidant activities of CMSPB80-1: DPPH• radical-scavenging activity (A); ABTS+• radical-scavenging activity (B). Values are the mean ± SD of three replicates.

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Fig. 8. Effect of CMSPB80-1 on RAW 264.7 cell viability (A); Effect of CMSPB80-1 on phagocytic rate of RAW 264.7 cells (B); Effect of CMSPB80-1 on production of NO (C); Effect of CMSPB80-1 on splenocyte proliferation (D). Data representative of three independent experiments were expressed as mean ± SD. *P < 0.05; **P < 0.01 vs. control group.

3.8. DPPH radicals scavenging assay

3.12. Production of nitric oxide (NO)

DPPH• is used as a stable free-radical to evaluate the scavenging ability of polysaccharides at A517 nm. The scavenging activity of CMSPB80-1 and Vc on the DPPH radicals were shown in Fig. 7A. The concentration ranged from 0.05 to 3.2 mg/mL of CMSPB80-1 and Vc was able to scavenge DPPH• radicals in a dose-dependent manner, which was consistent with (Zhang et al., 2015). The highest DPPH• radical scavenging rate of CMSPB80-1 was 47.45% (3.2 mg/mL)

In addition to phagocytosis, production of NO by macrophages also plays a key role in the process of bacterial killing (Khatua and Acharya, 2018). As shown in Fig. 8C, results showed that CMSPB80-1 did induce RAW 264.7 macrophages to release NO. Compared with control group, the production of NO increased significantly at concentrations of 62.5–500 μg/mL in a dose-dependent manner. 3.13. Proliferation of splenocyte lymphocytes

+

3.9. ABTS • radical scavenging assay

Lymphocyte proliferation is an index reflecting cellular immunity. Therefore, the determination of changes in splenocyte proliferation is an efficient method to investigate drug activity and active mechanisms. In the present study, in vitro cultured splenocytes obtained from KM mice were examined for the polysaccharides of CMSPB80-1 by comparing with the control and positive group (ConA). As illustrated in Fig. 8D, the results showed that CMSPB80-1 could obviously promote splenocyte lymphocytes proliferation at concentrations of 31.25–500 μg/mL when compared with control group.

+

The ABTS • radicals scavenging activity of CMSPB80-1 and Vc was measured in Fig. 7B. With the concentration of the samples increased, ABTS+• radical scavenging activity was enhanced. The highest ABTS+• radical scavenging rate for CMSPB80-1 was 49.58% (3.2 mg/mL). From the figure, the scavenging ability of CMSPB80-1 on ABTS+• radicals was concentration dependent, which was consistent with Wang et al. (2018a,Wang et al., 2018b. 3.10. Effects of CMSPB80-1 on RAW264.7 cell viability

4. Conclusion

The effects of CMSPB80-1on RAW264.7 cell viability, as shown in Fig. 8A, indicated no concentration of CMSPB80-1 obviously affected cell viability. Thus, the results indicated the polysaccharide of CMSPB80-1 was not cytotoxic to RAW264.7 cells at all tested concentration.

A novel heteropolysaccharide CMSPB80-1 from C. medica L. var. sarcodactylis was isolated, purified by alkali extraction and characterized the structure by UV spectrum, HPGPC analysis, monosaccharides analysis, FT-IR, methylation analysis, GC–MS and NMR spectroscopy. The HSQC, HMBC spectra and Congo red test implyed CMSPB80-1 exhibited an amorphous structure with a number of branches. Moreover, CMSPB80-1 exhibited antioxidant activity in a certain concentration range and obviously enhanced macrophage phagocytiosis of up-taking neutral red at all tested concentration. In addition, CMSPB80-1 could significantly promote the production of NO and splenocyte lymphocytes proliferation, which implied that CMSPB80-1 could be a good potential immunomodulatory agent for functional health food or assist in the treatment of some related diseases. As far as we know, this is the first report on the structures and functions of CMSPB80-1. However, the underlying mechanism of immune-enhancing activities and structureactivity relationship of the polysaccharide of CMSPB80-1 needs further elucidation.

3.11. Phagocytic activity It is well-known that macrophage activation is considered as one of the most important events in the immune response, and then further induce-activated innate immune response (Mosser and Edwards, 2008). In addition, enhancement of phagocytosis activity is accepted as one of the most distinguished features of macrophage activation as well (Chen et al., 2008). As shown in Fig. 8B, treatment with all concentration of CMSPB80-1 obviously augmented macrophage phagocytiosis. 8

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Declaration of Competing Interest

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