Structural characterisation and immunomodulatory activity of a neutral polysaccharide from Sambucus adnata Wall

Journal Pre-proofs Structural characterisation and immunomodulatory activity of a neutral polysaccharide from Sambucus adnata Wall Lei Yuan, Zheng-Chang Zhong, Yu Liu PII: DOI: Reference:

S0141-8130(19)36854-0 https://doi.org/10.1016/j.ijbiomac.2019.11.021 BIOMAC 13802

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

26 August 2019 22 October 2019 5 November 2019

Please cite this article as: L. Yuan, Z-C. Zhong, Y. Liu, Structural characterisation and immunomodulatory activity of a neutral polysaccharide from Sambucus adnata Wall, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.11.021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Elsevier B.V. All rights reserved.

Structural characterisation and immunomodulatory activity of a neutral polysaccharide from Sambucus adnata Wall. Lei Yuan a,b,*, Zheng-Chang Zhong b,c, Yu Liu c a

Centre of Physical & Chemic Analyses and Bio-tech, Tibet Agricultural & Animal Husbandry University, Linzhi 860000

Tibet, China b

Key Laboratory of Wildlife Resources Evaluation and Utilization in Tibet, Linzhi 860000 Tibet, China

c

Food Science College, Tibet Agricultural & Animal Husbandry University, Linzhi 860000 Tibet, China

Abstract A neutral polysaccharide, SPW-2, was purified from the leaves of Sambucus adnata Wall. using water extraction and alcohol precipitation, Sevage deproteination, ion exchange chromatography and gel filtration chromatography. This molecule had an average molecular weight of 7,040 Da and was composed of arabinose, xylose, mannose, glucose and galactose at a molar ratio of 1.5:0.5:1.2:5.0:1.8. The repetitive structural units of SPW-2 were deduced using methylation analysis and nuclear magnetic resonance (oneand two-dimensional) spectroscopy. In vitro immunological activity test showed that SPW-2 could induce the secretion of nitric oxide, interleukin-1β (IL-1β), IL-6 and tumour necrosis factor-alpha (TNF-α), and increase the mRNA expression level of inducible nitric oxide synthase (iNOS), IL-1β, IL-6 and TNF-α in macrophages. The data supported the notion that SPW-2 exerts an immunomodulatory effect by activating macrophages and enhancing the host immune system function, which enabled it to be used as a novel immunomodulator for application in the treatment of immunological diseases. Keywords: Sambucus adnata Wall., polysaccharide, structure, immunomodulatory activity

 *Corresponding author. E-mail address: [email protected] (L. Yuan) 1

1. Introduction China is the birthplace of traditional Chinese medicine, and polysaccharides are ubiquitous active ingredients in Chinese medicine. The immunomodulating activities of Chinese medicine polysaccharides are among the most important biological activities reported. Polysaccharides can promote lymphocyte proliferation; induce various cytokines; improve the production of interferon, interleukin (IL) and tumour necrosis factor (TNF); and activate macrophage, natural killer cells, lymphocytes and other immunocytes. These molecules also play a multifaceted regulatory role in immune function by improving specific and non-specific immune function in many ways [1–6]. Macrophages are the first line of defence of organisms. Their functions include protection against the invasion of pathogens, removal of foreign bodies and identification and killing of cancerous cells; they also play a primary role in maintaining internal environment stability, immune response and inflammatory response [7]. By presenting self-processed antigens to specific T cells, activated T cells further activate macrophages through molecules on cell membranes or ILs that they secreted to enhance their phagocytosis and release various active substances; these processes regulate the inflammatory response to monitor cellular immunity [8]. As an ideal cell model, macrophages are used to evaluate the immunoregulatory activity of active substances. Polysaccharides, which are active substances, can stimulate macrophages to secrete nitric oxide (NO), anti-TNF and IL expression, thereby improving the resistance of organisms to pathogenic microorganism invasion [9]. Sambucus adnata Wall. is a common Chinese folk medicine and one of the medicinal source plants of Tibetan medicine. This plant, which has a pungent and astringent taste, warms the internal organs inside the human body; it functions by dispelling wind, alleviating diuresis, promoting blood circulation and dredging collaterals. This medicine is mainly used for treating acute and chronic nephritis, rheumatic pain and other diseases. S. adnata Wall. is mainly distributed in the provinces of Shaanxi, Gansu, Qinghai, Sichuan, Guizhou and Yunnan and in the autonomous region of Ningxia; it is particularly abundant in Tibet and is mainly concentrated in the lower altitude areas of Southeastern Tibet or Southern Tibet [10]. Reports on the chemical constituents and biological activities of S. adnata Wall. are limited and have only focused on secondary metabolites and the quality control of medicinal materials [11]. The structural and activity analysis of polysaccharides from S. adnata Wall. has not been reported. In our previous study, one type of neutral polysaccharide, SPW-2, was purified from the leaves of S. adnata Wall. In this study, we used high-performance gel permeation chromatography (HPGPC), methylation, gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) to characterise the structure of SPW-2. We also investigated its immunomodulatory effect on macrophage RAW264.7 at the cellular and molecular levels. 2. Materials and Methods 2.1. Materials The samples, which consisted of leaves of S. adnata Wall. obtained from Juemugou, Linzhi City, Tibet, were dried and crushed. Sephacryl S-300HR and DEAE Sepharose Fast Flow were purchased from GE Healthcare (Stockholm, Sweden). Dextran standards [molecular weight (Mw) = 5,200, 11,600, 23,800, 48,600, 148,000, 273,000, 410,000, 668,000], monosaccharide standards (i.e. rhamnose, arabinose, galactose, glucose, mannose, xylose and fucose) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and foetal bovine serum were purchased from HyClone Laboratories (Logan, UT, USA). The Cell Counting Kit-8 (CCK-8) assay kit was purchased from 2

Biosharp Life Sciences (Heifei, China), and enzyme-linked immunosorbent assay kits for IL-1β, IL-6 and TNF-alpha (TNF-α) were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The NO assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.2. Extraction and purification of SPW-2 The leaves of S. adnata Wall. were extracted with petroleum ether at three times its volume and 95% industrial ethanol. Fat- and alcohol-soluble impurities were removed by refluxing three times for 2 h at each run. The pre-treated samples were heated for 114 min with a 1:26 material/liquid ratio of distilled water at 89°C for four times. The extracts were then combined and concentrated to 1/10 of the original volume, followed by precipitation in 80% (v/v) ethanol at 4°C for 24 h. The precipitate was collected through centrifugation at 4,000 rev/min for 10 min. Finally, crude polysaccharides (CSPs) were obtained by freeze-drying after the precipitate was dissolved in a small amount of distilled water. The CSPs were deproteinised using Sevage reagent (1-butanol/chloroform, v/v = 1:4) and further purified via anion-exchange chromatography using a DEAE-Sepharose Fast Flow column (3.5 × 40 cm) with ultrapure water as the eluent at a flow rate of 20 mL/10 min, followed by separation using a Sephacryl S-300HR gel column (2.5 × 100 cm) with 0.9% NaCl as the eluent at a flow rate of 10 mL/20 min to obtain the major peak. Finally, SPW-2 was obtained after concentration, dialysis and freeze-drying. 2.3. Structural characterisation of SPW-2 2.3.1. Determination of homogeneity and Mw of SPW-2 The homogeneity and average Mw of SPW-2 were determined via HPGPC combined with a refractive index detector equipped with a KS805-804-802 series gel column (7.8 × 300 mm; Japan) [12]. The concentration of SPW-2 solution was 2 mg/mL, and 20 μL of it was injected after filtering by using a 0.22-μm syringe filter. The Mw of SPW-2 was determined using 0.2 M NaCl as the eluent at a flow rate of 0.8 mL/min, and the temperature was maintained at 40°C. 2.3.2. Determination of total sugar and protein content Total sugars were estimated via the phenol–sulphuric acid method with glucose as the standard [13]. Total proteins were measured with the Bradford method [14] and using bovine serum albumin as the standard. 2.3.3. Monosaccharide composition analysis The monosaccharide composition analysis of SPW-2 was slightly modified in reference to the method described by Khatua [15]. First, 2 mg of SPW-2 was hydrolysed with 2 M CF3COOH (1 mL) at 120°C for 90 min, followed by evaporation to dryness. The hydrolysate was then reduced using 60 mg of NaBH4 for 8 h and acetylated with 1 mL of Ac2O/pyredine (11, v/v) at 100°C for 1 h. The alditol acetate derivatives were analysed using GCMS-QP2010 plus (Shimadzu, Kyoto, Japan) equipped with an Rxi-5Sil MS column (30 m × 0.25 mm I.D.). The temperature of the column oven was programmed as follows: initial temperature of 120°C, which was later raised to 250°C/min at 3°C/min and maintained for 5 min; detector temperature of 250°C, injector temperature of 250°C; and flow rate of He at 1 mL/min. The standard monosaccharides were treated and measured using the procedure described above. 2.3.4. Methylation analysis The methylation of polysaccharides was slightly modified in reference to the report of Mandal [16]. SPW-2 (3.0 mg) was dissolved in dried dimethyl sulphoxide (DMSO) (5.0 mL) and added with 20–30 mg of NaOH and 2 mL of CH3I for 60 min. The methylation reaction was terminated by adding 3 mL of ultrapure water. The methylated SPW-2 was hydrolysed with 2 M CF3COOH (1 mL) at 120°C for 90 min, followed by evaporation to dryness. The hydrolysate was then reduced using 60 mg of NaBH4 for 8 h and acetylated with 1 mL of Ac2O/pyredine (11, v/v) at 100°C for 1 h. The alditol acetate derivatives were 3

analysed using a GCMS-QP2010 plus (Shimadzu) equipped with an Rxi-5Sil MS column (30 m × 0.25 mm I.D.). The temperature of the column oven was programmed as follows: initial temperature of 120°C, which was raised to 250°C/min at 3°C/min and maintained for 5 min; detector temperature of 250°C; injector temperature of 250°C; and flow rate of He at 1 mL/min. 2.3.5. NMR spectroscopy SPW-2 (50 mg) was dissolved in D2O for replacement and freeze-dried. Then, the steps described above were repeated three times. The last freeze-dried sample was dissolved in D2O after P2O5 drying. All the NMR spectra of SPW-2, including 1H (500 MHz), 13C (126 MHz), 1H–1H correlation spectroscopy (COSY), heteronuclear single-quantum correlation spectroscopy (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC) and nuclear Overhauser effect spectroscopy (NOESY), were recorded using an AV500M NMR spectrometer (Bruker Instruments, Karlsruhe, Germany) at 25°C. The chemical shifts of NMR were processed and analysed using the MestReNova 6.1 software (Mestrelab Research, Escondido, CA, USA). 2.4. Immunological activity test of SPW-2 in vitro 2.4.1. Cell culture Mouse mononuclear macrophage RAW264.7 cells were obtained from Cell Resource Center of Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were cultured in DMEM containing 10% foetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C and 5% CO2 atmosphere. 2.4.2. Cytotoxicity assay of SPW-2 In vitro cytotoxicity was measured using CCK-8 assays. RAW264.7 cells (1 × 106 cells/mL) were inoculated in a 96-well plate. Next, different final concentrations of SPW-2 (100, 200, 500 and 1,000 μg/mL) were added. Wells without cells, SPW-2 and wells with 1.0 μg/mL LPS were treated as blank, negative control and positive control, respectively. After incubation for 24 h, the CCK-8 reagent (10 μL) was added into each well, and the mixtures were incubated for 1.5 h under the same condition. Absorbance was recorded at 450 nm. Cell viability (1) was calculated using the following equation: Cell viability (%) = [(ODSample − ODBlank)/(ODNegative − ODBlank)] × 100% (1) 2.4.3. Analysis of NO, IL-6, IL-1β and TNF-α in culture medium RAW264.7 cells (1 × 106 cells/mL) were inoculated into a 96-well plate. Next, different final concentrations of SPW-2 (100, 200, 500 and 1,000 μg/mL) were added. Wells without cells, SPW-2 and wells with 1.0 μg/mL LPS were treated as blank, negative control and positive control, respectively. The conditioned medium was collected for the analysis of NO, IL-6, IL-1β and TNF-α using the commercial kits according to the manufacturer’s instructions. 2.4.4. Real-time quantitative reverse transcription polymerase chain reaction The total RNA of RAW264.7 cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and synthesised into cDNA using a reverse transcription kit (TaKaRa, Tokyo, Japan). The CFX96 (BIO-RAD, USA) real-time polymerase chain reaction (PCR) system was used for the real-time quantitative amplification of genes. The primer sequences used were as follows: 3-phosphate dehydrogenase (GAPDH), forward: 5′-AGG TCG GTG TGA ACG GA TTT G-3′, reverse: 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′; iNOS: forward: 5′-GCC ACC AAC AAT GGC AAC AT -3′, reverse: 5′TCG ATG CAC AAC TGG GTG AA-3′; IL-6: forward: 5′-AGA CAA AGC CAG AGT CCT TCA G-3′, reverse: 5′-AGG AGA GCA TTG GAA ATT GGG-3′; IL-1β: forward: 5′-GAA ATG CCA CCT TTT GAC AGT GAT-3′, reverse: 5′-TTC TCC ACA GCC ACA ATG AGT-3′; TNF-α: forward: 5′-CAC CGT CAG CCG ATT TGC TA-3′ and reverse: 5′-TTG GGC AGA TTG ACC TCA GC-3′. The amplification 4

conditions were as follows: 95°C initial denaturation for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The GAPDH gene was selected as the internal reference gene, and the relative expression of the target genes was calculated using the 2–ΔΔct method. 2.5. Data analysis Data were expressed as mean ± standard deviation (SD) (n ≥ 3). The significance of the differences was estimated via one-way analysis of variance, followed by the least significant difference method using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Significant (p < 0.05) and highly significant differences (p < 0.01) compared with negative control were marked with single asterisk (*) and double asterisks (**), respectively. Meanwhile, significant (p < 0.05) and highly significant differences (p < 0.01) compared with positive control were marked with single pound (#) and double pound (##) signs, respectively. 3. Results and Discussion 3.1. Physicochemical properties of SPW-2 The neutral polysaccharide SPW-2 was purified using DEAE Sepharose Fast Flow and Sephacryl S-300HR (Fig. 1). SPW-2 as white powder with 91.83% sugar content and 2.94% protein content was eluted as a single symmetrical narrow peak on HPGPC (Fig. 2A), and its Mw is 7,040 Da. 3.2. Monosaccharide composition analysis of SPW-2 The hydrolysate of SPW-2 was acetylated and analysed via GC-MS. The results of alditol acetate derivatives of SPW-2 and monosaccharide standard are shown in Figure 2B. SPW-2 was mainly composed of arabinose, xylose, mannose, glucose and galactose with a molar ratio of 1.5:0.5:1.2:5.0:1.8. 3.3. Methylation analysis Methylation analysis, a classical method used in studying the primary structure of polysaccharides, is often used to analyse the connection mode of monosaccharide residues. In this method, the free –OH groups in the monosaccharide residues, which constitute the polysaccharide chain, are methylated completely under alkaline and non-aqueous conditions. The glycoside bond is then broken under acidic hydrolysis to obtain monosaccharide residues containing one or more –OH groups. The alditol acetate derivatives of methylated monosaccharide residues were obtained via acetylation derivatisation. Finally, the linking modes of polysaccharides were inferred according to the peak time, peak area and mass spectrum data of the derivatives obtained from GC-MS [17–19]. The fully methylated SPW-2 was hydrolysed with acid and analysed via GC-MS. The following 11 components—Araf-(1→, Xylp-(1→, Manp-(1→, Galp-(1→, →2)-Araf-(1→, →4)-Galp-(1→, →4)-Glcp-(1→, →3)-Galp-(1→, →2,4)-Galp-(1→, →4,6)-Glcp-(1→ and →3,6)-Manp-(1→—were found at a molar ratio of 0.6:0.4:0.5:0.4:0.5:1.8:3.8: 0.4:0.4:0.9:0.4 (Table 1). 3.4. NMR analysis NMR is a non-destructive method and an important tool for the primary structure analysis of polysaccharides. One- (1D) and two-dimensional (2D) NMR spectra are capable of distinguishing information on the following fields: polysaccharide structure, isomer type, monosaccharide type, glycoside bond position, connection mode and sequence. 1D NMR includes 1H NMR and 13C NMR, which can immediately provide information about the purity and anomeric configuration of the sample. 2D NMR mainly includes homonuclear chemical shift correlation spectra [COSY, NOESY and total correlation spectroscopy (TOCSY)] and heteronuclear chemical shift correlation spectra (HMQC, HSQC and HMBC), which can assign 1H NMR and 13C NMR peaks and provide structural information, such as anomeric configuration, glycoside bond type, linkage sequence and branched site in polysaccharides. Thus, this 5

method can determine the structure of polysaccharides, which other analytical techniques are unable to achieve [20,21]. In the 1H NMR spectrum (500 MHz, D2O) (Fig. 3A) of SPW-2, 11 signals of anomeric protons were found in the resonance region of SPW-2 and were located at δ = 5.34, 5.28, 5.18, 5.15, 5.02, 4.91, 4.71, 4.58, 4.53, 4.47 and 4.42. These measurements indicated the presence of at least five α configurations and six β configurations in the glycosidic bond of SPW-2. Sugar residues were designated as A to K according to the decreasing chemical shifts of anomeric proton. The 13C NMR spectrum (126 MHz, D2O) showed that the main anomeric carbon signals of SPW-2 were at δ = 110.54, 108.83, 108.67, 105.71, 104.75, 104.47, 103.85, 101.54, 101.34, 101.03 and 100.22 (Fig. 3B). To further analyse the structure of SPW-2, we examined the H–H COSY, HSQC, HMBC and NOESY profiles of SPW-2 by referring to related literature and methylation results on the same sugar residues (Fig. 4). The methylation results showed that the ratio of 4-α-D-Glcp-1→ (residue A) glycoside bond was the largest. Hence, the anomeric carbon and anomeric proton signals were 101.34 and 5.34 ppm, respectively. The chemical shifts of H-2 and H-3 were 3.57 and 3.89 ppm, respectively, which could be obtained by H–H COSY (Fig. 4A). The corresponding carbon spectra can be deduced from the HSQC maps (Fig. 4B) as δ72.96 and 74.77. According to the carbon spectrum analysis, δ78.80 and 61.90 were obtained for C-4 and C-6, respectively, and 3.59 and 3.77 ppm were obtained for H-4 and H-6a, respectively. According to the analysis of H–H COSY, the signal of H-4/5 was δ3.59/3.76, and that of C-5 was δ72.60. The corresponding cross signals were confirmed in HMBC (Fig. 4C) and NOESY (Fig. 4D) maps [21]. According to the methylation results, the 4)-β-D-Galp-(1→ (residue J) molar quantity was large, and the anomeric proton signal was 4.47 ppm. Meanwhile, the chemical shifts of H-2 and H-3 were 3.57 and 3.88 ppm, respectively, based on H–H COSY analysis. The corresponding carbon spectra were δ72.90 and 70.56. Incombination with the cross-peaks of C-2, C-3, H-4 and H-5 in HMBC, the signals of H-4 and H-5 were 4.05 and 3.81 ppm, respectively. Finally, H-6, C-4, C-5 and C-6 were assigned by combining information about H–H COSY, HSQC and NOESY [22]. Other glycosidic bond signals were assigned by combining data from H–H COSY, HSQC, HMBC and NOESY and by referring to similar rules and references as shown in Table 2 [23–33]. The linkage sequence was deduced from the HMBC spectrum. In HMBC, a correlation signal was found between C-1 (δ101.34) of residue A and its own H-4 δ3.59. This signal was ascribed to the correlation between H-1 and C-4 of inter-residue A. In addition, a correlation signal was found between H-1 and H-4 of inter-residue A, which further verified the existence of the links. The correlation signal between H-1 (δ5.34) of residue A and C-4 (δ78.14) of residue B indicated the existence of A–B links. The correlation signal between H-1 (δ5.34) of residue A and C-4 (δ78.10) of residue K indicated the existence of A–K links. The correlation signal between H-1 (δ5.28) of residue B and C-4 (δ77.84) of residue J indicated the existence of B–J links. In the NOESY spectrum, the relative peaks between H-1 (δ4.47) of residue J and H-4 (δ3.86) of residue K indicated the existence of J–K links. The relative peaks between H-1 (δ4.42) of K and H-4 (δ4.05) of residue J indicated the existence of K–J links. The relative signal between H-1 (δ4.47) of residue J and C-4 (δ78.80) of residue A indicated the existence of J–A links. In the HMBC spectrum, relative peaks were found among H-1 (δ5.18) of residue C and C-3 (δ85.71) of residue G, H-1 (δ4.91) of residue F, C-6 (δ67.54) of residue G, H-1 (δ4.71) of residue G and C-2 (δ77.08) of residue K, indicating the existence of C–G, F–G and G–K links. Relative signals were found among C-1 (δ108.67) of residue D and H-2 (δ4.04) of residue E, H-1 (δ5.02) of residue E and C-6 (δ67.88) of residue B, H-1 (δ4.58) of residue H, C-3 (δ78.57) of residue I, H-1 (δ4.53) of residue I and C-2 (δ77.08) of residue 6

K, indicating the existence of D–E, E–B, H–I and I–K links. The above links were further confirmed in the NOESY spectrum. On the basis of methylation analysis and NMR data, repetitive structure units of SPW-2 (Fig. 5) can be predicted (as discussed in the following subsections). 3.5. Immunomodulatory activity of SPW-2 3.5.1. Effect of SPW-2 on viability The cytotoxicity of SPW-2 against RAW264.7 macrophages was evaluated using the CCK-8 assay. The results shown in Figure 6 indicate that the negative control group and the SPW-2-treated groups had no significant differences (p > 0.01), indicating that SPW-2 (100–1,000 μg/mL) has no cytotoxicity towards RAW264.7 cells. 3.5.2. Effect of SPW-2 on NO and cytokine production Macrophages are an important component of the immune system, and they play a key role in host defence and acute inflammatory response [34]. Under the stimulation of external factors, these cells can be activated by simultaneously inducing NO and various cytokines, such as TNF-α, IL-1, IL-6 and IL-12 [35]. Therefore, the effect of polysaccharides on macrophages can be analysed by measuring the production of NO and related cytokines. Polysaccharides can stimulate macrophages to release various cytokines, such as NO, IL-1β, IL-6, IL-8, IL-12 and TNF-α, which mediate the body’s immune regulatory activity [36–39]. The effects of SPW-2 on NO and related cytokines in RAW264.7 cells are shown in Figure 7A. SPW-2 could significantly stimulate RAW264.7 cells to secrete NO at the concentration range of 200–1,000 μg/mL, indicating a substantial dose–effect relationship. When the concentration of SPW-2 was set at 1,000 μg/mL, the NO secretion was almost the same as that of the positive control. SPW-2 had no effect on the secretion of IL-1β in RAW264.7 cells at the concentration of 100–1,000 μg/mL but could increase the secretion of IL-6 and TNF-α. The secretion of IL-6 initially increased and then decreased. At the concentration of 200 μg/mL, the secretion of IL-6 reached the maximum at 7.65 times that of the control group and a significant level at 2.13 times that of the positive control group. The secretion of TNF-α was remarkably different from that of the negative control group; however, no differences were observed among the concentrations. 3.5.3. Effect of SPW-2 on mRNA expression of iNOS, IL-1β, IL-6 and TNF-α To further verify the effect of SPW-2 at different concentrations on NO and cytokine secretion in RAW264.7 cells, we determined the mRNA expression of iNOS, IL-1β, IL-6 and TNF-α via quantitative reverse transcription-PCR (qRT-PCR), and the relative expression was calculated using the 2-∆∆Ct method. The results are shown in Figure 7B. Compared with the negative control group, SPW-2 increased the relative expression of iNOS, IL-1β, IL-6 and TNF-α in RAW264.7 cells. A difference was observed in the mRNA expression of TNF-α but not in the TNF-α level at different concentrations compared with those of the control group. The mRNA expression levels of iNOS and IL-1β were dose-dependent, which had substantial differences at 200 and 1,000 μg/mL, respectively. 4. Conclusion A homogeneous neutral polysaccharide, SPW-2, was purified from the leaves of S. adnata Wall. Its structural features were systematically analysed, and its repeated structural units were predicted according to the structural features. The in vitro bioactivity tests showed that SPW-2 possessed notable immunomodulatory activities that may be mediated through the enhancement of NO, IL-6 and TNF-α production and the expression of iNOS, IL-1β, IL-6 and TNF-α. The results showed that SPW-2 could activate macrophages in normal state and had a strong immunoregulatory effect in vitro. The details on the 7

immune mechanisms and immune activity in vivo would be the subject of a continuing study, in order to establish a foundation for the application of SPW-2 as an immunomodulator. This study also provides useful information for the high-value utilisation of S. adnata Wall. and the development of related products in Tibet. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31560252). Conflicts of interests None. References [1] J. R. Chen, Z. Q. Yang, T. J. Hu, Z. T. Yan, T. X. Niu, L. Wang, D. A. Cui, M. Wang, Immunomodulatory activity in vitro and in vivo of polysaccharide from Potentilla anserine, Fitoterapia, 81 (8) (2010) 1117-1124. [2] T. Cárdenas-Reyna, C. Angulo, C. Guluarte, S. Hori-Oshima, M. Reyes-Becerril, In vitro immunostimulatory potential of fungal β-glucans in pacific red snapper (Lutjanus peru) cells, Dev. Comp. Immunol. 77 (2017) 350-358. [3] X. Zhang, C. Qi, Y. Guo, W. Zhou, Y. Zhang, Toll-like receptor 4-related immunostimulatory polysaccharides: primary structure, activity relationships, and possible interaction models, Carbohydr. Polym. 149 (2016) 186-206. [4] J. Meng, X. Hu, F. Shan, H. Hua, C. Lu, E. Wang, Z. Liang, Analysis of maturation of murine dendritic cells (DCs) induced by purified Ganoderma lucidum polysaccharides (GLPs), Int. J. Biol. Macromol. 49 (4) (2011) 693-699. [5] L. Xia, X. Liu, H. Guo, H. Zhang, J. Zhu, F. Ren, Partial characterization and immunomodulatory activity of polysaccharides from the stem of Dendrobium officinale (Tiepishihu) in vitro, J. Funct. Foods, 4 (1) (2012) 294-301. [6] Y. D. Yoon, S. B. Han, J. S. Kang, C. W. Lee, S. K. Park, H. S. Lee, J. S. Kang, H. M. Kim, Toll-like receptor 4-dependent activation of macrophages by polysaccharide isolated from the radix of Platycodon grandiflorum, Int. Immunopharmacol. 3 (13-14) (2003) 1873-1882. [7] S. Uthaisangsook, N. K. Day, S. L. Bahna, R. A. Good, S. Haraguchi, Innate immunity and its role against infections, Ann. Allerg. Asthma Im. 88 (3) (2002), 253-265. [8] I. A. Schepetkin, M. T. Quinn, Botanical polysaccharides: macrophage immunomodulation and therapeutic potential, Int. Immunopharmacol. 6 (3) (2006), 317-333. [9] M. Yin, Y. Zhang, H. Li, Advances in research on immunoregulation of macrophages by plant polysaccharides, Front. Immunol. (2019), 10. 145. 10.3389/fimmu.2019.00145. [10] Z. C. Ni, Economic plants of XIZANG(Tibet), Beijing: Beijing Science and Technology Press, (1990) 626-627. [11] H. B. Wu, Y. N. Zhao, D. M. Li, X. W. Lv, W. S. Wang, Studies on the Chemical Constituents from Sambucus adnata Wall, Nat. Prod. Res. Dev., 25 (2013) 345-348. (in Chinese) [12] J. Dai, Y. Wu, S. W. Chen, S. Zhu, H. P. Yin, M. Wang, J. Tang, Sugar compositional determination of polysaccharides from

Dunaliella

salina

by

modified

RP-HPLC

method

of

precolumn

derivatization

with

1-phenyl-3-methyl-5-pyrazolone, Carbohydr. Polym. 82 (3) (2010) 629-635. [13] M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (3) (1956) 350-356. [14] M. M. Bradford, A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1-2) (1976) 248-254. [15] S. Khatua, K. Acharya, Influence of extraction parameters on physico-chemical characters and antioxidant activity of water soluble polysaccharides from Macrocybe gigantea (massee) pegler & lodge, J. Food Sci. Tech. 53 (4) (2016) 8

1878-1888. [16] S. Mandal, R. Sarkar, P. Patra, C. K. Nandan, D. Das, S. K. Bhanja, S. S. Islam, Structural studies of a heteropolysaccharide (PS-I) isolated from hot water extract of fruits of Psidium guajava (Guava), Carbohyd Res, 344 (2009) 1365-1370. [17] M. H. Fischer, N. Yu, G. R. Gray, J. Ralph, L. Anderson, J. A. Marlett, The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk), Carbohyd. Res. 339 (11) (2004) 2009-2017. [18] J. Y. Yin, H. X. Lin, S. P. Nie, S. W. Cui, M. Y. Xie, Methylation and 2D NMR analysis of arabinoxylan from the seeds of Plantago asiatica L, Carbohydr. Polym. 88 (4) (2012) 1395-1401. [19] F. Wang, W. Wang, Y. Huang, Z. Liu, J. Zhang, Characterization of a novel polysaccharide purified from a herb of Cynomorium songaricum Rupr, Food Hydrocolloid. 47 (2015) 79-86. [20] D. Liu, Q. Sun,, J. Xu,, N. Li, J. Lin, S. Chen, F. Li, Purification, characterization, and bioactivities of a polysaccharide from mycelial fermentation of Bjerkandera fumosa, Carbohydr. Polym. 167 (2017) 115-122. [21] J. Liu, F. Shang, Z. Yang, M. Wu, J. Zhao, Structural analysis of a homogeneous polysaccharide from Achatina fulica, Int. J. Biol. Macromol. 98 (2017) 786-792. [22] J. Zhao, F. M. Zhang, X. Y. Liu, K. S. Ange, A. Q. Zhang, Q.H. Li, R. J. Linhardt, Isolation of a lectin binding rhamnogalacturonan-I containing pectic polysaccharide from pumpkin, Carbohydr. Polym. 163 (2017) 330-336. [23] P., Maity, M. Pattanayak, S. Maity, A. K. Nandi, I. K. Sen, B. Behera, T. K. Maiti, P. Mallick, S. R. Sikdar, S. S. Islam, A partially methylated mannogalactan from hybrid mushroom pfle 1p: purification, structural characterization, and study of immunoactivation. Carbohyd. Res. 395 (2014) 1-8. [24] C. K. Nandan, R. Sarkar, S. K. Bhanja, S.R. Sikdar, S.S. Islam, Isolation and characterization of polysaccharides of a hybrid mushroom (backcross mating between Pflovv12 and Volvariella volvacea), Carbohyd. Res. 346 (15) (2011) 2451-2456. [25] F. Li, S. H. Cui, X. Q. Zha, V. Bansal, Y. L. Jiang, M. N. Asghar, J. H. Wang, L. H. Pan, B. F. Xu, J. P. Luo, Structure and bioactivity of a polysaccharide extracted from protocorm-like bodies of Dendrobium huoshanense, Int. J. Biol. Macromol. 72 (2015) 664-672. [26] A. M. Gane, D. Craik, S. L. A. Munro, G. J. Howlett, A. E. Clarke, A. Bacic, Structural analysis of the carbohydrate moiety of arabinogalactan-proteins from stigmas and styles of Nicotiana alata, Carbohyd. Res. 277 (1) (1995) 67-85. [27] J. Kang, S. W. Cui, G. O. Phillips, J. Chen, Q. Guo, Q. Wang, New studies on gum ghatti (Anogeissus latifolia) part II. Structure characterization of an arabinogalactan from the gum by 1D, 2D NMR spectroscopy and methylation analysis, Food Hydrocolloid. 25 (8) (2011) 1991-1998. [28] J. Kang, S. W. Cui, G. O. Phillips, J. Chen, Q. Guo, Q. Wang, New studies on gum ghatti (Anogeissus latifolia) part III: Structure characterization of a globular polysaccharide fraction by 1D, 2D NMR spectroscopy and methylation analysis, Food Hydrocolloid. 25 (8) (2011) 1999-2007. [29] C. A. Tischer, M. Iacomini, R. Wagner, P. A. J. Gorin, New structural features of the polysaccharide from gum ghatti (Anogeissus latifola), Carbohyd. Res. 337 (21-23) (2002) 2205-2210. [30] A. K. Ojha, D. Maiti, K. Chandra, S. Mondal, D. Das, S. K. Roy, K. Ghosh, S. S. Islam, Structural assignment of a heteropolysaccharide isolated from the gum of Cochlospermum religiosum (Katira gum), Carbohyd. Res. 343 (7) (2008) 1222-1231. [31] D. Das, S. Mondal, S. K. Roy, D. Maiti, B. Bhunia, T. K. Maiti, S. S. Islam, Isolation and characterization of a heteropolysaccharide from the corm of Amorphophallus campanulatus, Carbohyd. Res. 344 (18) (2009) 2581-2585. [32] M. V. Svensson, X. Zhang, E. Huttunen, G. Widmalm, Structural studies of the capsular polysaccharide produced by Leuconostoc mesenteroides ssp. cremoris PIA2, Biomacromolecules, 12 (7) (2011), 2496-2501. [33] D. Pan, L. Wang, B. Hu, P. Zhou, Structural characterization and bioactivity evaluation of an acidic proteoglycan extract from Ganoderma lucidum fruiting bodies for PTP1B inhibition and anti-diabetes, Biopolymers, 101 (6) (2014), 9

613-623. [34] F. O. Martinez, L. Helming, S. Gordon, Alternative activation of macrophages: an immunologic functional perspective, Annu. Rev. Immunol. 27 (1) (2009) 451-483. [35] M. Rossol, H. Heine, U. Meusch, D. Quandt, C. Klein, M. J. Sweet, S. Hauschildt, LPS-induced cytokine production in human monocytes and macrophages, Crit. Rev. Immunol. 31 (5) (2011) 379-446. [36] C. Deng, J. Shang, H. Fu, J. Chen, H. Liu, J. Chen, Mechanism of the immunostimulatory activity by a polysaccharide from Dictyophora indusiata, Int. J. Biol. Macromol. 91 (2016) 752-759. [37] J. S. Lee, D. S. Kwon, K. R. Lee, J. M. Park, S. J. Ha, E. K. Hong, Mechanism of macrophage activation induced by polysaccharide from Cordyceps militaris culture broth, Carbohydr. Polym. 120 (2015) 29-37. [38] M. Wang, X. B. Yang, J. W. Zhao, C. J. Lu, W. Zhu, Structural characterization and macrophage immunomodulatory activity of a novel polysaccharide from Smilax glabra Roxb, Carbohydr. Polym. 156 (2017) 390-402. [39] D. Das, S. Maiti, T. K. Maiti, S. S. Islam, A new arabinoxylan from green leaves of Litsea glutinosa (Lauraeae): Structural and biological studies, Carbohydr. Polym. 92 (2) (2013) 1243-1248.

Tables and Figures: 10

Table 1 GC-MS data for the methylation analysis of SPW-2. Table 2 1H and 13C NMR chemical shifts of the SPW-2 in D2O. Figure 1 Isolation of polysaccharide present in the aqueous extract of S. adnata Wall. SPW was fractionated via ion-exchange chromatography on a DEAE-Sepharose Fast Flow column (A); SPW-2 was further purified via gel filtration chromatography on a Sephacryl S-300HR column(B). Figure 2 HPGPC profile and GC-MS total ion flow chromatography of SPW-2. Figure 3 1H NMR (A) and 13C NMR (B) spectra of SPW-2. Figure 4 H–H COSY(A), HSQC(B), HMBC(C) and NOESY(D) spectra of SPW-2 in D2O at 25 °C. Figure 5 Repeated structural unit of SPW-2. Figure 6 Effect of SPW-2 on the viability of RAW264.7 cells. Figure 7 Levels of NO, IL-1β, IL-6, TNF-α (A) and the mRNA expression of iNOS, IL-1β, IL-6 and TNF-α (B) of SPW-2 in RAW264.7 cells.

11

Figure 1



Figure 2



12

Figure 3.

Figure 4



13

Figure 5



Figure 6

14

Figure 7

15

Table 1 Peak No.

Methylated sugar

Mass fragments (m/z)

Molar ratios

Linkages

1

2,3,5-Me3-Araf

71, 87, 101, 117, 129, 145, 161

0.6

Araf-(1ൺ

2

2,3,4-Me3-Xylp

43, 101, 117, 161

0.4

Xylp-(1ൺ

3

3,5-Me2-Araf

71, 87, 101, 117, 129, 143, 161, 187

0.5

ൺ2)-Araf-(1ൺ

4

2,3,4,6-Me4-Manp

71, 87, 101, 117, 129, 145, 161, 205

0.5

Manp-(1ൺ

5

2,3,4,6-Me4-Galp

71, 87, 101, 117, 129, 145, 161, 205

0.4

Galp-(1ൺ

6

2,3,6-Me3-Galp

87, 99, 101, 113, 117, 129,131,161, 173, 233

1.8

ൺ4)-Galp-(1ൺ

7

2,3,6-Me3-Glcp

87, 99, 101, 113, 117, 129, 131, 161, 173, 233

3.8

ൺ4)-Glcp-(1ൺ

8

2,4,6-Me3-Galp

71, 87, 101, 117, 129, 161, 217, 233, 277

0.4

ൺ3)-Galp-(1ൺ

9

2,3-Me2-Glcp

71, 85, 87, 99, 101, 117, 127, 159, 161, 201

0.4

ൺ4,6)-Glcp-(1ൺ

10

3,6-Me2-Galp

71, 85, 87, 99, 113, 129, 143, 189, 233

0.9

ൺ2,4)-Galp-(1ൺ

11

2,4-Me2-Manp

71, 87, 99, 101, 117, 129, 139, 159, 189

0.4

ൺ3,6)-Manp-(1ൺ

        

16

      Table 2 Glycosyl residues

A: →4)-α-D-Glcp-(1→

B: →4,6)-α-D-Glcp-(1→

C: T-α-Xylp→

D: α-L-Araf-1→

E: →2)-α-L-Araf-1→

F: β-D-Manp-(1→

G: →3,6)-β-D-Manp-(1→

H: β-D-Galp-(1→

I: →3)-β-D-Galp-(1→

J: →4)-β-D-Galp-(1→

K: →2,4)-β-D-Galp-(1→

H1

H2

H3

H4

H5a

H5b/H6a

C1

C2

C3

C4

C5

C6

5.34

3.57

3.89

3.59

3.76

3.77

101.34

72.96

74.77

78.80

72.60

61.90

5.28

3.56

3.88

3.73

4.05

3.77

101.03

71.24

72.62

78.14

74.28

67.88

5.18

3.74

3.70

3.86

3.66

3.77

110.54

78.10

81.40

84.60

62.60

5.15

3.88

4.22

3.69

3.95

108.67

78.20

83.29

81.33

61.73

5.02

4.04

4.17

3.67

3.97

108.83

85.37

83.57

80.33

61.73

4.91

3.48

3.73

3.99

3.55

4.03

100.22

72.60

71.20

70.90

74.60

61.90

4.71

3.49

3.75

3.62

3.97

3.85

101.50

72.60

85.71

71.09

75.40

67.54

4.58

3.63

4.08

3.88

3.70

3.77

105.71

71.20

71.50

70.60

74.30

61.90

4.53

3.49

3.64

3.31

3.76

3.61

104.47

72.63

78.57

72.61

70.77

62.82

4.47

3.57

3.88

4.05

3.81

3.97

103.85

72.90

70.56

77.84

72.90

61.93

4.42

3.48

3.75

3.86

3.66

3.85

104.75

77.08

74.70

78.10

75.30

61.79

  

17

H6b

3.71

4.08

4.02

4.02

3.62

3.55

3.71

3.49

3.62

3.75

Highlights: · · · ·

One neutral polysaccharide SPW-2 was isolated from the leaves of S. adnata Wall.. SPW-2 composed of arabinose, xylose, mannose, glucose and galactose with Mw of 7040Da. The structure characterizations of SPW-2 were analyzed. SPW-2 exerted high RAW264.7 macrophage stimulating activity.

18

*UDSKLFDODEVWUDFW

H–H COSY(A), HSQC(B), HMBC(C) and NOESY(D) spectra of SPW-2 in D2O at 25 °C.



19