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Structure characterization of two functional polysaccharides from Polygonum multiflorum and its immunomodulatory
Accepted Manuscript Structure characterization of two functional polysaccharides from Polygonum multiflorum and its immunomodulatory
Qing Zhang, Yi Xu, Junjiang Lv, Mengxia Cheng, Ying Wu, Kun Cao, Xiaofeng Zhang, Xiuni Mou, Qi Fan PII: DOI: Reference:
S0141-8130(17)34381-7 doi:10.1016/j.ijbiomac.2018.02.064 BIOMAC 9118
To appear in: Received date: Revised date: Accepted date:
9 November 2017 2 February 2018 11 February 2018
Please cite this article as: Qing Zhang, Yi Xu, Junjiang Lv, Mengxia Cheng, Ying Wu, Kun Cao, Xiaofeng Zhang, Xiuni Mou, Qi Fan , Structure characterization of two functional polysaccharides from Polygonum multiflorum and its immunomodulatory. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.02.064
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ACCEPTED MANUSCRIPT Title page Structure Characterization of two Functional Polysaccharides from Polygonum multiflorum and its Immunomodulatory Qing Zhang
a,d
Kun Cao a,b
Xiaofeng Zhang a,b
a,b,c1
Junjiang Lv
a,b
Mengxia Cheng
a,b
Ying Wu
a,b
Xiuni Mou a,b Qi Fan a,b
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Yi Xu
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a School of Chemistry and Chemical Engineering, Chongqing University, Chongqing,
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400030, P.R.China
b Defense Key Disciplines Lab of Novel Micro-nano Devices and System
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Technology, Chongqing University,Chongqing, 400030, P.R.China
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c International R & D center of Micro-nano Systems and New Materials Technology, Chongqing University, Chongqing, 400030, P.R.China
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d Chongqing Institute of Food and Drug Control, Chongqing, 401121, P.R.China
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Abstract
Two purified polysaccharides named WPMP-1 and WPMP-2 were obtained from
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Polygonum multiflorum with an average molecular weight of 2.04 kDa and 92.13 kDa, respectively. Structural characterization revealed that WPMP-1was supposed to be a glucan composed of 1,4-linked α-D-Glcp, 1,4,6-linked α-D-Glcp, and terminal
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α-D-Glcp. WPMP-2 contained a complex branched structre formed by 1,2-linked α-D-Rhap, 1,2,4-linked α-D-Rhap, 1,3,5- linked α-L-Ara, 1,5- linked α-L-Araf, terminal α-L-Araf, 1,4-linked β-D-Galp, 1,4-linked α-D-GalpA, and 4-linked α-D-GalpA. In vitro, WPMPs were of activation effect on splenocyte and
1
Corresponding author: Yi Xu, School of Chemistry and Chemical Engineering, Chongqing
University, Chongqing, 400030, P.R.China Phone: (+86) 023-65111022; e-mail: [email protected]
ACCEPTED MANUSCRIPT macrophages,
and
could
also
protect
immunocyte
against
5-Fu
induced
immunosupression by restoring the proliferation rate, phagocytic index and cytokines secretion level. Moreover, the acid polysaccharide WPMP-2 exhibited better immunomodulatory activity than neutral polysaccharide WPMP-1, which indicated that the immunomodulatory activity of WPMPs were positively associated with
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higher content of uronic acid, percentage of rhamnose, arabinose and galactose, and branching degree. These findings could promote the polysaccharides from Polygonum
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multiflorum to be attractive functional food supplement and immunomodulatory
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adjuvant.
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Keywords: polysaccharides; Polygonum multiflorum; structure characterization; immunomodulatory activity.
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1. Introduction
Natural polysaccharides have been widely used as important immunomodulatory
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supplements in health products, such as polysaccharides from Ganoderma,[1,2]
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Lepidium meyenii,[3] Cordyceps,[4] Platycladus orientalis,[5] and Angelica root[6] were all have been proved effective in immunocompetence. These natural polysaccharides could also potentially improving tumor treatment by protecting immune cells and cells
against
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hematopoietic
chemotheraphy-induced
cellular
damage,[7]
and
stimulating the antibody and cytokines production (IL - 2, IL - 6 , TNF-α, et al.)[8-10]
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in the case of immunosuppression caused by chemotherapy. Polygonum multiflorum is the dryed roots of Polygonum multiflorum Thunb, one of well-known traditional Chinese herbal medicine, which has been widely used in treatment of hyperlipemia, anti-aging, antibiosis, strengthening immunity, improving cardiovascular function, et al. In the past, stibene glucoside as one of the main active components of Polygonum multiflorum has been studied thoroughly. However, only a few researchers had paid attention on the antioxidant activity, [11] antitumor activity[12] and immnunomodulatory activity[13] of water-extracted polysaccharides of Polygonum
ACCEPTED MANUSCRIPT multiflorum. Also, there was few report about the chain structure of WPMPs, and lack structural basis for the further structure-activity relationship analysis. In this study, two water-soluble purified polysaccharides named WPMP-1 and WPMP-2 were prepared from Polygonum multiflorum and characterized by HPLC analysis, methylation analysis, FTIR and NMR analysis. The immunomodulatory
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activity of these functional polysaccharides were evaluated by determining proliferation of splenocytes, phagocytosis of macrophages, and level of cytokines. In
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additon, the primary correlations between immunomodulatory activity and structural
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features were discussed. The results will be helpful for potentially commercial use of the Polygonum multiflorum polysaccharides as functional food ingredients or
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immunoregulatory adjuvants.
2. Experimental
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2.1 Materials
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The Polygonum multiflorum (dried products) were purchased from a local commercial market in Chongqing, China. DEAE-52 and sephadex G100 were purchased from Whatman (Maidstone, Kent, UK); The enzyme-linked immunosorbent assay (ELISA)
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kits for interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd., China, and all other analytic
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chemical reagents were purchased from Kelong Chemical (Chengdu, China). 2.2 Animals for experiments KM mice (female, 20.0 ± 2.0 g) were purchased from the Laboratory Animal Center of Chongqing Medical University, Chongqing, China (the license number: SCXK (Yu) 2007-0001). During the experiment the mice were kept under standard laboratory conditions. All the experimental procedures were approved by the Animal Care Committee of Chongqing University. 2.3 Preparation of WPMPs
ACCEPTED MANUSCRIPT Extraction and purification of polysaccharides of Polygonum multiflorum (WPMPs) was processed as described by Lv.[11] Briefly, the crude Polygonum multiflorum polysaccharides (WPMP) was obtained following the steps of water extraction, deproteination, decolorization, alcohol precipitation and lyophilization. Then, a total of 2.5g WPMP was separated into two fractions by DEAE cellulose-52 anion
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exchange column chromatography (4.6 × 40 cm), and eluted with deionized water and 2.0 M NaCl solutions at a flow rate of 1.0 mL/min. After dialyzed for 48 h, and
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lyophilized, the two fractions were further purified by Sephadex G-100 chromatography (2.6 × 40 cm), eluted with deionized water at a flow rate of 0.5
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mL/min. Finally, two purified polysaccharide WPMP-1 and WPMP-2 were prepared, respectively.
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2.4 Structure Characterization of WPMPs
Molecular weight determination. The homogeneity and molecular weight
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distribution of WPMPs were determined by high performance gel permeation chromatography (HPGPC) on an Agilent 1100 HPLC system equipped with KS-802
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and KS- 804 column in series and a differential refractive index detector (RI-
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101SHODEX). Column temperature was 30 °C. The samples were eluted with 0.2 M NaCl aqueous solution at a flow rate of 0.5 mL/min. A series of glucan (P-5, P-10, P-20, P-50, P-100, P-200, P-400 and P-800) had been chosen as the calibration
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standards. The retention time (tR) was obtained to calculate the weight-averaged
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molecular weight (Mw) of WPMPs based on the following equation: 𝑙𝑜𝑔𝑀𝑤 = −0.353𝑡𝑅 + 7.50
(1)
Infrared Spectrum Analysis. The Fourier transform infrared (FT-IR) spectrum was obtained by using a Shimadzu FT-IR spectrometer (Shimadzu Spectrum 400 FT-IR, JP) at the range of 4000-400 cm−1 . Monosaccharide chromatographic
composition method
was
Analysis. applied
to
The
high
simultaneous
performance determinate
liquid seven
monosaccharides (glucose, rhamnose, mannose, arabinose, galactose, xylose, and galacturonic acid) by pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP).[14] In detail, a total of 10.0 mg of WPMP-1 or WPMP-2 was hydrolyzed by
ACCEPTED MANUSCRIPT TFA (2.0 M) at 120 °C for 8 h in a sealed ampoule. After cooling to room temperature, the reaction mixture was centrifuged and the supernatant was collected and lyophilized. Then the hydrolyzed sample or monosaccharide standard aqueous solution was mixed with 1.0 mL of aqueous NaOH (0.3 M) and 1.0 mL of PMP methanol solution (0.5 M) thoroughly, and incubated at 70 °C for 90 min. After
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cooled and neutralized with HCl (0.3 M), the resulting solution was extracted with chloroform to remove the excess reagents (PMP). Finally, the aqueous layer was
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filtered through a 0.45µm membrane, analyzed by HPLC: Agilent 1260 HPLC system equipped with a ZORBAX SB-C18 (250 mm × 4.6 mm, column temperature: 30 °C),
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the wavelength of detection was 250 nm, elution was carried out with a mixture of acetonitrile and phosphate buffer (0.06 M, pH 6.8) in a ratio of 16: 84 (v/v, %) at a
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flow rate of 1.0 mL/min, and the injection value was 20 µL.
Methylation analysis. According to the methods from literature
[15]
with some
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modification. The lyophilized WPMP-1 or WPMP-2 (15.0 mg) was dissolved in 5.0 mL anhydrous DMSO contain anhydrous sodium hydroxide powder (240.0 mg) and
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sonicated for 20 min at 60 °C. Then 3.0 mL methyl iodide was added into mixture
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with stirring in 15 min, after reaction for 1 hour in dark, the reaction was stopped by the addition of 6 mL distilled water. The obtained solution was dialyzed against distilled water for 48 h and then lyophilized. This procedure was repeated until the
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infrared absorption of hydroxy completely disappeared at 3400cm-1. The methylated polysaccharide was extracted with chloroform for three times and evaporated to
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dryness, the dryed methylated polysaccharide was hydrolyzed by TFA (2.0M) at 120 °C for 8 h. The hydrolysates were reduced with 70.0 mg NaBH4 at 50 °C for 24 h, and acetylated with acetic anhydride (0.5 mL) and pyridine (0.5 mL) at 100 °C for 2 h. The acetic anhydride was decomposed by 1.0 mL distilled water, and evaporated to dryness. The acetylated derivatives was dissolved in chloroform and analyzed by gas chromatography (GC) coupled with mass spectrometry (MS) (Agilent, USA) using a HP-5 capillary column (30 m × 0.25 mm, 0.25 µm thickness, 150-200 °C at 2 °C /min, and then 200-280 °C at 5 °C /min).
ACCEPTED MANUSCRIPT NMR analysis. About 30.0 mg of WPMP-1 or WPMP-2 was dissolved in 0.55 mL of D2O in a NMR tube and then the
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C NMR, 1H NMR, HSQC and HMBC spectra
were recorded on a NMR apparatus (Agilent, 600Hz) at 298 K. 2.5 Immunomodulatory Activity of WPMPs The potential immunomodulatory effect of WPMPs was evaluated by determining its
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potential activition on proliferation of splenocytes and phagocytosis of peritoneal macrophages, and protection on splenocytes and peritoneal macrophages against 5-Fu
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induced immunosuppression. The preparation of splenocytes and peritoneal macrophages, and cell culture process were operated refer to our previously study. [16]
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Briefly, the aqueous treatment solution of WPMPs were first prepared in a serial concentrations (20, 50, 100, 300 and 500 μg/mL). Splenocytes and peritoneal
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macrophages were cultured in 96-well plates for 24h, respectively. After incubated with treatment solution of WPMPs for another 48 h, the cell viability was measured
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based on MTT method, the absorbance at 570 nm was obtained to calculate the cell viability of splenocytes based on the equation-2: 𝐴 570𝑛𝑚(𝑠𝑎𝑚𝑝𝑙𝑒)
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𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = 𝐴
570𝑛𝑚(𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
× 100%
(2)
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Where the cell viability of splenocytes from control group was calculated as 100%. The phagocytosis index was measured based on neutral red experiment,
[17]
the
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absorbance at 540 nm was obtained to calculate phagocytosis of peritoneal
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macrophages based on the equation-3: 𝑃ℎ𝑎𝑔𝑜𝑐𝑦𝑡𝑜𝑠𝑖𝑠 𝑖𝑛𝑑𝑒𝑥 =
𝐴540𝑛𝑚(𝑠𝑎𝑚𝑝𝑙𝑒) 𝐴540𝑛𝑚(𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
× 100%
(3)
Where the phagocytosis index of peritoneal macrophages from normal control group was calculated as 100%. The cytokines (IL-2 and TNF-α) existed in the culture supernatants of splenocytes and peritoneal macrophages were measured by ELISA kits according to the manufacturers’ manner. All experiments were performed in triplicate. Statistical analysis
ACCEPTED MANUSCRIPT Mean data values are presented with their deviation(mean ± SD). Analysis of variance (ANOVA) was followed by T-test for pairwise comparison. Statistical significance was defined as p < 0.05 for all tests.
3. Results and discussion
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3.1 Extraction and Purification of polysaccharides
The crude polysaccharides (WPMP) was obtained after refluxing extraction,
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deproteinization and decolorization from Polygonum multiflorum with a yield of
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3.8%. Then, WPMP was separated by DEAE-52 anion-exchange chromatography, and further purified sequentially by Sephadex G-100 gel filtration chromatography.
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As a result, two purified polysaccharides named WPMP-1 and WPMP-2 were isolated from the crude polysaccharides (figure S1). Both WPMP-1 and WPMP-2 showed one
single
symmetrical
peak
on
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only
high
performance
gel-permeation
chromatography (figure S2), which indicated that they were homogeneous
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polysaccharides. the Mw of WPMP-1 and WPMP-2 were determined to be 2.04 KDa
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and 92.13 KDa, respectively. During the preparation of polysaccharides, an improved decoloring method was established based on MPLC (Medium Pressure column Chromatography) system with a reversed phase MCI GEL CHP 20P column (4.6 × 40
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cm). The commercial MCI GEL CHP 20P is a kind of microsphere packing (φ75-150µm) of polymethacrylate skeleton. Its separation mechanism was integration
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of reversed-phase chromatographic separation and gel filtration, which was good match for separation of pigments or micromolecules from crude polysaccharide according to the differences in polarity and molecular weight. In detail, the polysaccharides extraction was injection into the chromatographic column and eluted by triple column volumes deionized water and methanol in sequence at a flow rate of 5.0 mL/min, the water elution was collected within a short time and lyophilized to obtain the decolored polysacccharides. Compared with the traditional decoloring methods such as activated carbon adsorption and hydrogen peroxide oxidation, the loss of polysaccharides and decoloring time of the new method were significantly
ACCEPTED MANUSCRIPT reduced, and there was no possibility of residual organic solvent, carbonous residues and structure destruction. 3.2 Characterization of WPMP-1 and WPMP-2 Monosaccharide composition. As shown in figure 1, WPMP-1 was suggested to be glucan, while WPMP-2 contained rhamnose, galacturonic acid, galactose, arabinose.
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The molar percentages of monosaccharides were calculated according to the external standard method. As a result, the monosaccharide molar ratio of WPMP-2 was
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rhamnose (1.65): galacturonic acid (1.00): galactose (1.08): arabinose(3.40) , and trace amount of glucose. It was revealed that the monosaccharide composition of
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WPMP-1 was quite different from that of WPMP-2.
Figure 1. HPLC chromatograms of PMP derivatives of mixed monosaccharide standards (a), WPMP-1 (b) and WPMP-2 (c).
FT-IR spectrum. The functional groups and fingerprint regions of WPMP-1 and WPMP-2 were characterized by FT-IR spectrometry. The characteristic bands in the regions of 3400, 2925 and 1420cm−1 attributing to hydroxyl stretching vibration, C-H stretching vibration and C-H bending vibration, respectively, which were
ACCEPTED MANUSCRIPT characteristic absorption bands of polysaccharide. The three absorption bands at 1000-1200 cm−1 suggested the existence of C-O stretching vibrations of glycosidic bonds and pyranoid rings.[18] In the FT-IR spectrum of WPMP-1, the stretching peak in the regions of 938, 842,and 765cm−1 suggested WPMP-1 was a α-D-glucopyranose derivative.[19] In the FT-IR spectrum of WPMP-2, the stretching peak at 1743 cm−1
β-pyranoside linkage at 890 cm−1 was also observed. [21]
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suggested the presence of carboxyl group,[20] A characteristic absorption of
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Methylation analysis. The GC–MS chromatogram results of WPMP-1 and WPMP-2
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were shown in figure S4 and figure S5. According to the data of total ion chromatogram and literatures,[20-22] the analysis result of the glycosidic linkage was
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shown in Table 1. The molar ratio of glycoside residues was estimated from the peak areas.
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Three glycoside residues were identified in WPMP-1, namely, (1→4)-linked-Glcp, (1→4,6)-linked-Glcp, and terminal Glcp, with molar ratio of 3.70: 1.00: 1.10. it was indicated that WPMP-1 was a glucan with →4)- Glcp-1→ as backbone. The main
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branching point was at O-6 of (1→4,6)-linked-Glcp, and the ratio between terminal
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units and branching points was 1:1, indicating that the number of terminal units was approximately equal to the number of branching points in WPMP-1.
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The result of GC-MS of WPMP-2 showed that there were at least six recognizable methylated glycoside residues, namely terminal Araf, (1→2)-linked-Rhap, (1→2,4)-
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linked-Rhap, (1→5)-linked- Araf, (1→3, 5)-linked- Araf, and (1→4)-linked-Galp, with molar ratio of 0.82: 2.33: 0.81: 3.86: 1.00: 2.07. The main branching points were at (1→2,4)- linked-Rhap and (1→3, 5)-linked- Araf. The ratio between terminal units and branching points was approximately 1:2, indicating that WPMP-2 has multi-branching structure. It was worth reminding that there was no signal of galacturonic acid residue been observed, due to WPMP-2 had not been reduced before methylation. However, the signal of galacturonic acid residue existed in the HPLC analysis. Table 1. Methylation analysis and glycosidic linkage composition of WPMP-1 and WPMP-2
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Terminal Glcp
1.10
43,58,71,101,117,129,145,161,205
1,4-linked Glcp
3.70
43,58,71,87,101,113,117,129,131,143,161,173,233
1,4,6-linked Glcp
1.00
43,58,85,101,117,127,142,159,201,261
Terminal Araf
0.82
43,59,71,87,101,102,118,129,161
1,5-linked- Araf
3.86
43,87101,117,129,189
1,3,5-linked- Araf
1.00
43,85,99,117,127,129,159,201
1,2-linked-Rhap
2.33
43,88,89,100,101,115,130,131,161,190
1,2,4-linked-Rhap
0.81
43,87,101,129,143,189,203
1,4-linked-Galp
2.07
45,71,87,99,102,113,118,129,131,162,173,233
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Mass fragments(m/z)
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WPMP-2
Molar ratio
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WPMP-1
Type of linkage
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NMR analysis. Linkages between glycosyl residues in WPMP-1 and WPMP-2 were further investigated by NMR analyses, including 1H NMR and
C NMR, with the
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assistance of HSQC and HMBC spectra.
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As shown in Figure 2, three signals appeared in the anomeric region(1H is from 4.3 to 5.9 ppm, whereas
13
C is in the range of 92−112 ppm) of both the 1H and
13
C NMR
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spectra of WPMP-1, and three cross peaks were presented in the anomeric region of
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the HSQC spectrum, suggesting the presence of three different linkage patterns. According to the results of linkage analysis and the literature,[19] The anomeric proton
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signals at 5.25, 5.28, and 4.85 ppm (figure 2a) and the anomeric carbon signals at δ 99.48, 99.53, and 98.5 ppm (figure 2b) were corresponded to H-1 and C-1 of (residue A): →4)-α-D-Glcp-(1→, (residue B): →4,6)-α-D-Glcp- (1→, and (residue C):
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α-D-Glcp(1→, respectively. The molar ratio of the residues ((A + B)/C) was estimated as 5.35: 1 from peak area of the integration of the H-1 signal of A, B, and C residues, and it was in agreement with that of methylation analysis (appropriately 5.80: 1). Obviously, WPMP-1 could be confirmed to consist of repeating units →4)-α-D-Glcp-(1→4)-α-D-Glcp- (1→ as backbone with branches at O-6 position of →4,6)-α-D-Glcp- (1→ (average of 5-6 glucose units). Furthermore, all of the chemical shifts of WPMP-1 were completely assigned and listed in table S1.
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ACCEPTED MANUSCRIPT
Figure 2. 1H (a), 13C (b), HSQC (c), and HMBC (d) NMR spectra of WPMP-1 in D2O solution at
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25 °C.
The NMR spectra of WPMP-2 were shown in figure 3. Signals of the anomeric proton at 4.86 ppm was indicative of the β-anomeric configuration.[23] Signals of the
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anomeric proton at 1.14 ppm and anomeric carbon at 16.5 ppm elucidated the
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presence of rhamnose residue.[24,25] Signals in the region between 170.8 and 175.2 ppm in the 13C NMR corroborated the presence of the uronic acids, while the signal at
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170.8 ppm was attributed to the signal peak of carboxylic carbon of uronic acid, the signal at 175.2 ppm could be assigned to the unesterified carboxylic carbon, and the
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signals at δ1.95 ppm (1H NMR) and δ 20.99 ppm (13C NMR) were corresponded to the -CH3 groups of α- GalpA on O-2/3.[20] As shown in figure 3c, eight cross peaks were presented in the anomeric region of the HSQC spectrum, suggesting the presence of eight different linkage patterns. According to the results of linkage analysis and the literature,[20,24,26-29] the anomeric proton signals at 4.86, 4.99, 5.00, 5.01, 5.10, 5.11, 5.20 and 5.66 ppm in the 1H spectrum (figure 3a), and the related anomeric carbon signals at 100.13, 107.31, 98.78, 98.94, 99.06, 107.12, 92.11 and 106.86 ppm (figure 3b) were corresponded to H-1 and C-1 of 1,4-linked-β-D-Galp, 1,5-linked-α-L-Araf,
1,2,4-linked-α-
D-Rhap,
1,2-linked-α-D-Rhap,
ACCEPTED MANUSCRIPT 1,4-linked-α-D-GalpA, terminal α-L-Araf, 4-linked-α-D-GalpA and 1,3,5-linkedα-L-Araf residues, respectively. The other chemical shift assignments of 1H and their corresponding
13
C were identified and are summarized in Table S2 from the above
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spectra according to the literature data.
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Figure 3. 1H (a), 13C (b), HSQC (c), and HMBC (d) NMR spectra of WPMP-2 in D2O solution at 25 °C
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On the basis of the results of HPLC, methylation and NMR analysis, as shown in figure 4, the proposed partial structure of WPMP-1 and WPMP-2 were designed. It
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was indicated that there were great differences in monosaccharide composition, branching degree, and content of uronic acid between WPMP-1and WPMP-2.
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3.3 Immunomodulatory Activity of WPMPs
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Figure 4. Proposed partial structure of WPMP-1 (a) and WPMP-2 (b).
The proliferation of splenocytes and phagocytosis of peritoneal macrophages were
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taken as parameters to evaluate the potential immunomodulatory activity of WPMPs in vitro, because splenocytes and macrophages are the most important immunocytes of immunity system, the splenocytes proliferation is crucial to cellular and humoral
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immune responses.[30]
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immune responses, and activated macrophages are essential to the innate and adaptive
Activation effect of WPMPs on splenocytes and peritoneal macrophages in vitro.
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As shown in figure 5a, WPMPs increased the cell viability of splenocytes in a dose-dependent manner at concentration from 20 to 500 µg/mL compared to the
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normal control group (##p < 0.05). It was illustrated that all the WPMPs samples could promote the proliferation of splenocytes. Comparing the promotion effect of WPMP, WPMP-1 and WPMP-2, WPMP-2 showed the strongest active effect on proliferation of splenocytes, followed by WPMP and WPMP-1. As shown in figure 5b, WPMPs could enhance the phagocytic capacity of peritoneal macrophages. Compared to the control group, 20 µg/mL WPMP and WPMP-2 could significantly enhanced the uptake of neutral red (p < 0.05), while that of WPMP-1 was 100µg/mL, and the enhancement was stronger with the increasing of concentration of polysaccharides. What’s more, the effect of WPMP-2 (500 µg/mL)
ACCEPTED MANUSCRIPT was higher than that of LPS group (p < 0.01). The result indicated that the acidic purified polysaccharides WPMP-2 demonstrated much stronger activation effect on
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phagocytosis of peritoneal macrophages than WPMP-1.
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Figure 5. Effects of different concentrations of WPMPs on(a) proliferation of splenocytes, the cell viability of untreated splenocyte was calculated as 100%, (b) Phagocytosis of peritoneal macrophages. The phagocytosis index of untreated peritoneal macrophages was calculated as 100%. The groups with RPMI 1640 medium and LPS (10 μg/mL) were the normal and model control group, respectively. The result repre-sented the mean ± SD (n = 3), #p < 0.05 and
##
p<
0.01 compared with the normal control group; ★p < 0.05 and ★★p < 0.01 compared with the LPS group.
ACCEPTED MANUSCRIPT Cytoprotective effect of WPMPs on 5-Fu treated splenocytes and peritoneal macrophages. 5-Fu is a representative anticancer agent but with severe side effects such as immunosuppression, etc.[7,31] As shown in figure 6, the proliferation and phagocytosis of splenocytes/peritoneal macrophages treated with 5-Fu (20 μg/mL) were markedly decreased compared with the normal control group (p < 0.01).
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Conversely, the proliferation of splenocytes and phagocytosis of peritoneal macrophage treated with mixture polysaccharides and 5-Fu were all significantly
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recovered compared with these of splenocytes/peritoneal macrophage treated with 5-Fu alone (p < 0.05), and the degree of recovery increased with the dose of
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polysaccharides increased. It was indicated that WPMPs could protect immunocytes aganist 5-Fu induced cellular damage and inhibition. And WPMP-2 demonstrated
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much stronger protective effect on splenocytes/peritoneal macrophages than WPMP
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and WPMP-1.
ACCEPTED MANUSCRIPT Figure 6. Cytoprotective effects of WPMPs on 5-Fu treated (a) splenocytes, the cell viability of normal group was calculated as 100%, (b)peritoneal macrophages. Phagocytosis index of normal group was calculated as 100%. The groups with RPMI 1640 medium and 5-Fu (20μg/mL) were the normal and model control group, respectively. The result represented the mean ± SD (n = 3), p < 0.01 compared with the normal control group; ★p < 0.05 and ★★p < 0.01 compared with the
##
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model control group.
Effects of WPMPs on IL-2 and TNF-α secretion. To further study the
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immunomodulatory activity of WPMPs, IL-2 and TNF-α levels in the supernatants of
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cell cultures of 5-Fu treated splenocytes and macrophages in vitro were measured by ELISA, because cytokines (IL-2, TNF-α, etc.) are signaling molecules that control
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homeostasis of the organism by regulating cell differentiation, proliferation, and apoptosis, as well as defense functions.[17, 32-35].
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As shown in figure 7, 5-Fu demonstrated strong inhibiting effect on IL-2 (962.6±37.3ng/L) and TNF-α (637.9±24.5ng/L) secretion compared with the normal control group (1175.3±34.6ng/L and 776.1±35.9ng/L, respectivly) (p < 0.01).
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However, there was a dose-dependent upregulation of IL-2 secretion in the groups of
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combinative administration of 5-Fu and WPMPs (figure 7a). What’s more, WPMPs showed significantly activation effect on the expression of IL-2 (≥1257.9±64.2 ng/L)
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at high concentrations (500 μg/mL), which was visible higher than that of normal control group (p < 0.01). The similar increasing of TNF-α secretion in the supernatant
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of macrophage cultures after combined administration of 5-Fu and WPMPs was also observed (figure 7b). Particularly, the TNF-α level of WPMP-2 (≥100 μg/mL) was significantly higher compared to normal control group (p < 0.01),and WPMP-2 showed much more excellent TNF-α secretion upregulate activity than the others. It was indicated that WPMPs could play a role of immunomodulator by stimulate the secretion of cytokines.
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ACCEPTED MANUSCRIPT
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Figure 7. Effects of the WPMPs on splenocytes IL-2 (a) and macrophages TNF-α (b) production.
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RPMI 1640 medium and 5-Fu (20 μg/mL) were the normal and model control group, respectively. The result represented the mean ± SD (n = 3). ##p < 0.01 compared with the normal control group; p < 0.05 and ★★p < 0.01 compared with the model control group.
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★
3.4 Structure-activity relationship of WPMPs
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It is well known that the molecular weights, monosaccharide composition, functional groups, branching degrees, and glycosidic linkages were some of the structural features identified as determining or contributing to the polysaccharides immunostimulatory activity.[36] Generally, a high branching degree is benefit to the formation of triple helices in D-glucans which is positively associated to immunostimulatory activity.[37,38] The degree of branching and uronic acids have been regarded as important roles in antioxidant activity,[24,39,40] and many acid polysaccharides were proved to be active in antitumor and immunomodulatory.[27,39,40] The presence of acetyl and sulfate groups of mannans, galactans, and fucoidans are
ACCEPTED MANUSCRIPT crucial structural features for immunostimulatory activity.[39,40] In some cases, molecular weight could be not as relevant as other structural features,[36] etc. However, these structural features may have different relevance depending on the type of polysaccharides and their combination may impact the resulting activity in different ways.
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The potential immunomodulatory activity of WPMPs could be comprehensively evaluated by combining the activation and cytoprotection activity on immunocytes. It
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was indicated that the immunomodulatory activity of WPMP-2 was markedly better than that of WPMP-1 and WPMP. Since WPMP was the mixture of WPMP-1and
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WPMP-2, the bioactivity of WPMP could be regarded as the aggregated results of WPMP-1and WPMP-2. Considering the molecular weight, content of uronic acid, and
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degree of branching of WPMP-2 were all higer than those of WPMP-1, and according to the above literatures on structure-activity relationship of polysaccharides, the
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presence of uronic acid group, and higher branching degree and molecular weight were suggested to be the positive characteristics for the better immunostimulatory
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4. Conclusions
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activity.
In this study, two completely different homogeneous polysaccharides named
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WPMP-1 and WPMP-2 were purified from Polygonum multiflorum and their structural features and immunomodulatory activity have been investigated. The
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molecular weight of two homogeneous polysaccharides were 2.04kDa and 92.13kDa, respectively. WPMP-1 was constituted by 1,4-linked α-D-Glcp as backbone, with a side branch at O-6 position of 1,4,6-linked α-D-Glcp. WPMP-2 was constituted by 1,2-linked α-D-Rhap, 1,2,4-linked α-D-Rhap, and 1,4-linked α-D-GalpA as the backbone, with side chain of either arabinan (1,3,5-linked α-L-Araf, 1,5-linked α-L-Araf, terminal α-L-Araf) or galactooligosaccharides (1,4-linked β-D-Galp, 4-linked
α-D-GalpA)
Immunomodulation
on
assays
O-2/4
position
of
indicated
that
WPMPs
1,2,4-linked
α-D-Rhap.
possessed
significant
immunomodulatory activity by promoting proliferation of splenocytes and activating
ACCEPTED MANUSCRIPT phagocytosis of peritoneal macrophages, and also protecting immunocytes against 5-Fu induced immunosuppression. Moreover, due to the presence of uronic acid group, and higher branching degree and molecular weight, WPMP-2 doing much better both in activation and cytoprotection than WPMP-1. It was illustrated that polysaccharides presented in Polygonum multiflorum were definitely worth further
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exploring, and utilized as immunomodulator contribute to food and pharmaceutical industries, particularly, WPMP-2 could be more efficacious in lessening
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chemotherapy induced immunosuppression.
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Acknowledgements
This research work was supported by Innovative Project on Designing and Screening
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Drug Candidates of Chongqing (cstc2015zdcy-ztzx120003), the Key Project of Frontier and Basic Research of Chongqing Science and Technology Committee
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(cstc2015jcyjBX0010), and Innovation Project Program of Chongqing Science and Technology Committee (cstc2015shmszx00014).
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Notes
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The authors declare no competing financial interest.
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Qing Zhang, Yi Xu, Junjiang Lv, Mengxia Cheng, Ying Wu, Kun Cao, Xiaofeng Zhang, Xiuni Mou, Qi Fan PII: DOI: Reference:
S0141-8130(17)34381-7 doi:10.1016/j.ijbiomac.2018.02.064 BIOMAC 9118
To appear in: Received date: Revised date: Accepted date:
9 November 2017 2 February 2018 11 February 2018
Please cite this article as: Qing Zhang, Yi Xu, Junjiang Lv, Mengxia Cheng, Ying Wu, Kun Cao, Xiaofeng Zhang, Xiuni Mou, Qi Fan , Structure characterization of two functional polysaccharides from Polygonum multiflorum and its immunomodulatory. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.02.064
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ACCEPTED MANUSCRIPT Title page Structure Characterization of two Functional Polysaccharides from Polygonum multiflorum and its Immunomodulatory Qing Zhang
a,d
Kun Cao a,b
Xiaofeng Zhang a,b
a,b,c1
Junjiang Lv
a,b
Mengxia Cheng
a,b
Ying Wu
a,b
Xiuni Mou a,b Qi Fan a,b
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Yi Xu
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a School of Chemistry and Chemical Engineering, Chongqing University, Chongqing,
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400030, P.R.China
b Defense Key Disciplines Lab of Novel Micro-nano Devices and System
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Technology, Chongqing University,Chongqing, 400030, P.R.China
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c International R & D center of Micro-nano Systems and New Materials Technology, Chongqing University, Chongqing, 400030, P.R.China
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d Chongqing Institute of Food and Drug Control, Chongqing, 401121, P.R.China
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Abstract
Two purified polysaccharides named WPMP-1 and WPMP-2 were obtained from
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Polygonum multiflorum with an average molecular weight of 2.04 kDa and 92.13 kDa, respectively. Structural characterization revealed that WPMP-1was supposed to be a glucan composed of 1,4-linked α-D-Glcp, 1,4,6-linked α-D-Glcp, and terminal
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α-D-Glcp. WPMP-2 contained a complex branched structre formed by 1,2-linked α-D-Rhap, 1,2,4-linked α-D-Rhap, 1,3,5- linked α-L-Ara, 1,5- linked α-L-Araf, terminal α-L-Araf, 1,4-linked β-D-Galp, 1,4-linked α-D-GalpA, and 4-linked α-D-GalpA. In vitro, WPMPs were of activation effect on splenocyte and
1
Corresponding author: Yi Xu, School of Chemistry and Chemical Engineering, Chongqing
University, Chongqing, 400030, P.R.China Phone: (+86) 023-65111022; e-mail: [email protected]
ACCEPTED MANUSCRIPT macrophages,
and
could
also
protect
immunocyte
against
5-Fu
induced
immunosupression by restoring the proliferation rate, phagocytic index and cytokines secretion level. Moreover, the acid polysaccharide WPMP-2 exhibited better immunomodulatory activity than neutral polysaccharide WPMP-1, which indicated that the immunomodulatory activity of WPMPs were positively associated with
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higher content of uronic acid, percentage of rhamnose, arabinose and galactose, and branching degree. These findings could promote the polysaccharides from Polygonum
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multiflorum to be attractive functional food supplement and immunomodulatory
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adjuvant.
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Keywords: polysaccharides; Polygonum multiflorum; structure characterization; immunomodulatory activity.
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1. Introduction
Natural polysaccharides have been widely used as important immunomodulatory
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supplements in health products, such as polysaccharides from Ganoderma,[1,2]
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Lepidium meyenii,[3] Cordyceps,[4] Platycladus orientalis,[5] and Angelica root[6] were all have been proved effective in immunocompetence. These natural polysaccharides could also potentially improving tumor treatment by protecting immune cells and cells
against
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hematopoietic
chemotheraphy-induced
cellular
damage,[7]
and
stimulating the antibody and cytokines production (IL - 2, IL - 6 , TNF-α, et al.)[8-10]
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in the case of immunosuppression caused by chemotherapy. Polygonum multiflorum is the dryed roots of Polygonum multiflorum Thunb, one of well-known traditional Chinese herbal medicine, which has been widely used in treatment of hyperlipemia, anti-aging, antibiosis, strengthening immunity, improving cardiovascular function, et al. In the past, stibene glucoside as one of the main active components of Polygonum multiflorum has been studied thoroughly. However, only a few researchers had paid attention on the antioxidant activity, [11] antitumor activity[12] and immnunomodulatory activity[13] of water-extracted polysaccharides of Polygonum
ACCEPTED MANUSCRIPT multiflorum. Also, there was few report about the chain structure of WPMPs, and lack structural basis for the further structure-activity relationship analysis. In this study, two water-soluble purified polysaccharides named WPMP-1 and WPMP-2 were prepared from Polygonum multiflorum and characterized by HPLC analysis, methylation analysis, FTIR and NMR analysis. The immunomodulatory
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activity of these functional polysaccharides were evaluated by determining proliferation of splenocytes, phagocytosis of macrophages, and level of cytokines. In
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additon, the primary correlations between immunomodulatory activity and structural
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features were discussed. The results will be helpful for potentially commercial use of the Polygonum multiflorum polysaccharides as functional food ingredients or
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immunoregulatory adjuvants.
2. Experimental
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2.1 Materials
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The Polygonum multiflorum (dried products) were purchased from a local commercial market in Chongqing, China. DEAE-52 and sephadex G100 were purchased from Whatman (Maidstone, Kent, UK); The enzyme-linked immunosorbent assay (ELISA)
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kits for interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd., China, and all other analytic
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chemical reagents were purchased from Kelong Chemical (Chengdu, China). 2.2 Animals for experiments KM mice (female, 20.0 ± 2.0 g) were purchased from the Laboratory Animal Center of Chongqing Medical University, Chongqing, China (the license number: SCXK (Yu) 2007-0001). During the experiment the mice were kept under standard laboratory conditions. All the experimental procedures were approved by the Animal Care Committee of Chongqing University. 2.3 Preparation of WPMPs
ACCEPTED MANUSCRIPT Extraction and purification of polysaccharides of Polygonum multiflorum (WPMPs) was processed as described by Lv.[11] Briefly, the crude Polygonum multiflorum polysaccharides (WPMP) was obtained following the steps of water extraction, deproteination, decolorization, alcohol precipitation and lyophilization. Then, a total of 2.5g WPMP was separated into two fractions by DEAE cellulose-52 anion
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exchange column chromatography (4.6 × 40 cm), and eluted with deionized water and 2.0 M NaCl solutions at a flow rate of 1.0 mL/min. After dialyzed for 48 h, and
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lyophilized, the two fractions were further purified by Sephadex G-100 chromatography (2.6 × 40 cm), eluted with deionized water at a flow rate of 0.5
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mL/min. Finally, two purified polysaccharide WPMP-1 and WPMP-2 were prepared, respectively.
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2.4 Structure Characterization of WPMPs
Molecular weight determination. The homogeneity and molecular weight
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distribution of WPMPs were determined by high performance gel permeation chromatography (HPGPC) on an Agilent 1100 HPLC system equipped with KS-802
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and KS- 804 column in series and a differential refractive index detector (RI-
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101SHODEX). Column temperature was 30 °C. The samples were eluted with 0.2 M NaCl aqueous solution at a flow rate of 0.5 mL/min. A series of glucan (P-5, P-10, P-20, P-50, P-100, P-200, P-400 and P-800) had been chosen as the calibration
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standards. The retention time (tR) was obtained to calculate the weight-averaged
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molecular weight (Mw) of WPMPs based on the following equation: 𝑙𝑜𝑔𝑀𝑤 = −0.353𝑡𝑅 + 7.50
(1)
Infrared Spectrum Analysis. The Fourier transform infrared (FT-IR) spectrum was obtained by using a Shimadzu FT-IR spectrometer (Shimadzu Spectrum 400 FT-IR, JP) at the range of 4000-400 cm−1 . Monosaccharide chromatographic
composition method
was
Analysis. applied
to
The
high
simultaneous
performance determinate
liquid seven
monosaccharides (glucose, rhamnose, mannose, arabinose, galactose, xylose, and galacturonic acid) by pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP).[14] In detail, a total of 10.0 mg of WPMP-1 or WPMP-2 was hydrolyzed by
ACCEPTED MANUSCRIPT TFA (2.0 M) at 120 °C for 8 h in a sealed ampoule. After cooling to room temperature, the reaction mixture was centrifuged and the supernatant was collected and lyophilized. Then the hydrolyzed sample or monosaccharide standard aqueous solution was mixed with 1.0 mL of aqueous NaOH (0.3 M) and 1.0 mL of PMP methanol solution (0.5 M) thoroughly, and incubated at 70 °C for 90 min. After
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cooled and neutralized with HCl (0.3 M), the resulting solution was extracted with chloroform to remove the excess reagents (PMP). Finally, the aqueous layer was
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filtered through a 0.45µm membrane, analyzed by HPLC: Agilent 1260 HPLC system equipped with a ZORBAX SB-C18 (250 mm × 4.6 mm, column temperature: 30 °C),
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the wavelength of detection was 250 nm, elution was carried out with a mixture of acetonitrile and phosphate buffer (0.06 M, pH 6.8) in a ratio of 16: 84 (v/v, %) at a
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flow rate of 1.0 mL/min, and the injection value was 20 µL.
Methylation analysis. According to the methods from literature
[15]
with some
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modification. The lyophilized WPMP-1 or WPMP-2 (15.0 mg) was dissolved in 5.0 mL anhydrous DMSO contain anhydrous sodium hydroxide powder (240.0 mg) and
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sonicated for 20 min at 60 °C. Then 3.0 mL methyl iodide was added into mixture
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with stirring in 15 min, after reaction for 1 hour in dark, the reaction was stopped by the addition of 6 mL distilled water. The obtained solution was dialyzed against distilled water for 48 h and then lyophilized. This procedure was repeated until the
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infrared absorption of hydroxy completely disappeared at 3400cm-1. The methylated polysaccharide was extracted with chloroform for three times and evaporated to
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dryness, the dryed methylated polysaccharide was hydrolyzed by TFA (2.0M) at 120 °C for 8 h. The hydrolysates were reduced with 70.0 mg NaBH4 at 50 °C for 24 h, and acetylated with acetic anhydride (0.5 mL) and pyridine (0.5 mL) at 100 °C for 2 h. The acetic anhydride was decomposed by 1.0 mL distilled water, and evaporated to dryness. The acetylated derivatives was dissolved in chloroform and analyzed by gas chromatography (GC) coupled with mass spectrometry (MS) (Agilent, USA) using a HP-5 capillary column (30 m × 0.25 mm, 0.25 µm thickness, 150-200 °C at 2 °C /min, and then 200-280 °C at 5 °C /min).
ACCEPTED MANUSCRIPT NMR analysis. About 30.0 mg of WPMP-1 or WPMP-2 was dissolved in 0.55 mL of D2O in a NMR tube and then the
13
C NMR, 1H NMR, HSQC and HMBC spectra
were recorded on a NMR apparatus (Agilent, 600Hz) at 298 K. 2.5 Immunomodulatory Activity of WPMPs The potential immunomodulatory effect of WPMPs was evaluated by determining its
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potential activition on proliferation of splenocytes and phagocytosis of peritoneal macrophages, and protection on splenocytes and peritoneal macrophages against 5-Fu
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induced immunosuppression. The preparation of splenocytes and peritoneal macrophages, and cell culture process were operated refer to our previously study. [16]
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Briefly, the aqueous treatment solution of WPMPs were first prepared in a serial concentrations (20, 50, 100, 300 and 500 μg/mL). Splenocytes and peritoneal
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macrophages were cultured in 96-well plates for 24h, respectively. After incubated with treatment solution of WPMPs for another 48 h, the cell viability was measured
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based on MTT method, the absorbance at 570 nm was obtained to calculate the cell viability of splenocytes based on the equation-2: 𝐴 570𝑛𝑚(𝑠𝑎𝑚𝑝𝑙𝑒)
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𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = 𝐴
570𝑛𝑚(𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
× 100%
(2)
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Where the cell viability of splenocytes from control group was calculated as 100%. The phagocytosis index was measured based on neutral red experiment,
[17]
the
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absorbance at 540 nm was obtained to calculate phagocytosis of peritoneal
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macrophages based on the equation-3: 𝑃ℎ𝑎𝑔𝑜𝑐𝑦𝑡𝑜𝑠𝑖𝑠 𝑖𝑛𝑑𝑒𝑥 =
𝐴540𝑛𝑚(𝑠𝑎𝑚𝑝𝑙𝑒) 𝐴540𝑛𝑚(𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
× 100%
(3)
Where the phagocytosis index of peritoneal macrophages from normal control group was calculated as 100%. The cytokines (IL-2 and TNF-α) existed in the culture supernatants of splenocytes and peritoneal macrophages were measured by ELISA kits according to the manufacturers’ manner. All experiments were performed in triplicate. Statistical analysis
ACCEPTED MANUSCRIPT Mean data values are presented with their deviation(mean ± SD). Analysis of variance (ANOVA) was followed by T-test for pairwise comparison. Statistical significance was defined as p < 0.05 for all tests.
3. Results and discussion
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3.1 Extraction and Purification of polysaccharides
The crude polysaccharides (WPMP) was obtained after refluxing extraction,
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deproteinization and decolorization from Polygonum multiflorum with a yield of
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3.8%. Then, WPMP was separated by DEAE-52 anion-exchange chromatography, and further purified sequentially by Sephadex G-100 gel filtration chromatography.
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As a result, two purified polysaccharides named WPMP-1 and WPMP-2 were isolated from the crude polysaccharides (figure S1). Both WPMP-1 and WPMP-2 showed one
single
symmetrical
peak
on
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only
high
performance
gel-permeation
chromatography (figure S2), which indicated that they were homogeneous
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polysaccharides. the Mw of WPMP-1 and WPMP-2 were determined to be 2.04 KDa
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and 92.13 KDa, respectively. During the preparation of polysaccharides, an improved decoloring method was established based on MPLC (Medium Pressure column Chromatography) system with a reversed phase MCI GEL CHP 20P column (4.6 × 40
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cm). The commercial MCI GEL CHP 20P is a kind of microsphere packing (φ75-150µm) of polymethacrylate skeleton. Its separation mechanism was integration
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of reversed-phase chromatographic separation and gel filtration, which was good match for separation of pigments or micromolecules from crude polysaccharide according to the differences in polarity and molecular weight. In detail, the polysaccharides extraction was injection into the chromatographic column and eluted by triple column volumes deionized water and methanol in sequence at a flow rate of 5.0 mL/min, the water elution was collected within a short time and lyophilized to obtain the decolored polysacccharides. Compared with the traditional decoloring methods such as activated carbon adsorption and hydrogen peroxide oxidation, the loss of polysaccharides and decoloring time of the new method were significantly
ACCEPTED MANUSCRIPT reduced, and there was no possibility of residual organic solvent, carbonous residues and structure destruction. 3.2 Characterization of WPMP-1 and WPMP-2 Monosaccharide composition. As shown in figure 1, WPMP-1 was suggested to be glucan, while WPMP-2 contained rhamnose, galacturonic acid, galactose, arabinose.
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The molar percentages of monosaccharides were calculated according to the external standard method. As a result, the monosaccharide molar ratio of WPMP-2 was
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rhamnose (1.65): galacturonic acid (1.00): galactose (1.08): arabinose(3.40) , and trace amount of glucose. It was revealed that the monosaccharide composition of
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WPMP-1 was quite different from that of WPMP-2.
Figure 1. HPLC chromatograms of PMP derivatives of mixed monosaccharide standards (a), WPMP-1 (b) and WPMP-2 (c).
FT-IR spectrum. The functional groups and fingerprint regions of WPMP-1 and WPMP-2 were characterized by FT-IR spectrometry. The characteristic bands in the regions of 3400, 2925 and 1420cm−1 attributing to hydroxyl stretching vibration, C-H stretching vibration and C-H bending vibration, respectively, which were
ACCEPTED MANUSCRIPT characteristic absorption bands of polysaccharide. The three absorption bands at 1000-1200 cm−1 suggested the existence of C-O stretching vibrations of glycosidic bonds and pyranoid rings.[18] In the FT-IR spectrum of WPMP-1, the stretching peak in the regions of 938, 842,and 765cm−1 suggested WPMP-1 was a α-D-glucopyranose derivative.[19] In the FT-IR spectrum of WPMP-2, the stretching peak at 1743 cm−1
β-pyranoside linkage at 890 cm−1 was also observed. [21]
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suggested the presence of carboxyl group,[20] A characteristic absorption of
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Methylation analysis. The GC–MS chromatogram results of WPMP-1 and WPMP-2
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were shown in figure S4 and figure S5. According to the data of total ion chromatogram and literatures,[20-22] the analysis result of the glycosidic linkage was
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shown in Table 1. The molar ratio of glycoside residues was estimated from the peak areas.
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Three glycoside residues were identified in WPMP-1, namely, (1→4)-linked-Glcp, (1→4,6)-linked-Glcp, and terminal Glcp, with molar ratio of 3.70: 1.00: 1.10. it was indicated that WPMP-1 was a glucan with →4)- Glcp-1→ as backbone. The main
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branching point was at O-6 of (1→4,6)-linked-Glcp, and the ratio between terminal
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units and branching points was 1:1, indicating that the number of terminal units was approximately equal to the number of branching points in WPMP-1.
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The result of GC-MS of WPMP-2 showed that there were at least six recognizable methylated glycoside residues, namely terminal Araf, (1→2)-linked-Rhap, (1→2,4)-
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linked-Rhap, (1→5)-linked- Araf, (1→3, 5)-linked- Araf, and (1→4)-linked-Galp, with molar ratio of 0.82: 2.33: 0.81: 3.86: 1.00: 2.07. The main branching points were at (1→2,4)- linked-Rhap and (1→3, 5)-linked- Araf. The ratio between terminal units and branching points was approximately 1:2, indicating that WPMP-2 has multi-branching structure. It was worth reminding that there was no signal of galacturonic acid residue been observed, due to WPMP-2 had not been reduced before methylation. However, the signal of galacturonic acid residue existed in the HPLC analysis. Table 1. Methylation analysis and glycosidic linkage composition of WPMP-1 and WPMP-2
ACCEPTED MANUSCRIPT
Terminal Glcp
1.10
43,58,71,101,117,129,145,161,205
1,4-linked Glcp
3.70
43,58,71,87,101,113,117,129,131,143,161,173,233
1,4,6-linked Glcp
1.00
43,58,85,101,117,127,142,159,201,261
Terminal Araf
0.82
43,59,71,87,101,102,118,129,161
1,5-linked- Araf
3.86
43,87101,117,129,189
1,3,5-linked- Araf
1.00
43,85,99,117,127,129,159,201
1,2-linked-Rhap
2.33
43,88,89,100,101,115,130,131,161,190
1,2,4-linked-Rhap
0.81
43,87,101,129,143,189,203
1,4-linked-Galp
2.07
45,71,87,99,102,113,118,129,131,162,173,233
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Mass fragments(m/z)
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WPMP-2
Molar ratio
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WPMP-1
Type of linkage
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NMR analysis. Linkages between glycosyl residues in WPMP-1 and WPMP-2 were further investigated by NMR analyses, including 1H NMR and
C NMR, with the
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assistance of HSQC and HMBC spectra.
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As shown in Figure 2, three signals appeared in the anomeric region(1H is from 4.3 to 5.9 ppm, whereas
13
C is in the range of 92−112 ppm) of both the 1H and
13
C NMR
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spectra of WPMP-1, and three cross peaks were presented in the anomeric region of
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the HSQC spectrum, suggesting the presence of three different linkage patterns. According to the results of linkage analysis and the literature,[19] The anomeric proton
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signals at 5.25, 5.28, and 4.85 ppm (figure 2a) and the anomeric carbon signals at δ 99.48, 99.53, and 98.5 ppm (figure 2b) were corresponded to H-1 and C-1 of (residue A): →4)-α-D-Glcp-(1→, (residue B): →4,6)-α-D-Glcp- (1→, and (residue C):
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α-D-Glcp(1→, respectively. The molar ratio of the residues ((A + B)/C) was estimated as 5.35: 1 from peak area of the integration of the H-1 signal of A, B, and C residues, and it was in agreement with that of methylation analysis (appropriately 5.80: 1). Obviously, WPMP-1 could be confirmed to consist of repeating units →4)-α-D-Glcp-(1→4)-α-D-Glcp- (1→ as backbone with branches at O-6 position of →4,6)-α-D-Glcp- (1→ (average of 5-6 glucose units). Furthermore, all of the chemical shifts of WPMP-1 were completely assigned and listed in table S1.
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ACCEPTED MANUSCRIPT
Figure 2. 1H (a), 13C (b), HSQC (c), and HMBC (d) NMR spectra of WPMP-1 in D2O solution at
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25 °C.
The NMR spectra of WPMP-2 were shown in figure 3. Signals of the anomeric proton at 4.86 ppm was indicative of the β-anomeric configuration.[23] Signals of the
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anomeric proton at 1.14 ppm and anomeric carbon at 16.5 ppm elucidated the
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presence of rhamnose residue.[24,25] Signals in the region between 170.8 and 175.2 ppm in the 13C NMR corroborated the presence of the uronic acids, while the signal at
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170.8 ppm was attributed to the signal peak of carboxylic carbon of uronic acid, the signal at 175.2 ppm could be assigned to the unesterified carboxylic carbon, and the
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signals at δ1.95 ppm (1H NMR) and δ 20.99 ppm (13C NMR) were corresponded to the -CH3 groups of α- GalpA on O-2/3.[20] As shown in figure 3c, eight cross peaks were presented in the anomeric region of the HSQC spectrum, suggesting the presence of eight different linkage patterns. According to the results of linkage analysis and the literature,[20,24,26-29] the anomeric proton signals at 4.86, 4.99, 5.00, 5.01, 5.10, 5.11, 5.20 and 5.66 ppm in the 1H spectrum (figure 3a), and the related anomeric carbon signals at 100.13, 107.31, 98.78, 98.94, 99.06, 107.12, 92.11 and 106.86 ppm (figure 3b) were corresponded to H-1 and C-1 of 1,4-linked-β-D-Galp, 1,5-linked-α-L-Araf,
1,2,4-linked-α-
D-Rhap,
1,2-linked-α-D-Rhap,
ACCEPTED MANUSCRIPT 1,4-linked-α-D-GalpA, terminal α-L-Araf, 4-linked-α-D-GalpA and 1,3,5-linkedα-L-Araf residues, respectively. The other chemical shift assignments of 1H and their corresponding
13
C were identified and are summarized in Table S2 from the above
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spectra according to the literature data.
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Figure 3. 1H (a), 13C (b), HSQC (c), and HMBC (d) NMR spectra of WPMP-2 in D2O solution at 25 °C
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On the basis of the results of HPLC, methylation and NMR analysis, as shown in figure 4, the proposed partial structure of WPMP-1 and WPMP-2 were designed. It
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was indicated that there were great differences in monosaccharide composition, branching degree, and content of uronic acid between WPMP-1and WPMP-2.
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ACCEPTED MANUSCRIPT
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3.3 Immunomodulatory Activity of WPMPs
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Figure 4. Proposed partial structure of WPMP-1 (a) and WPMP-2 (b).
The proliferation of splenocytes and phagocytosis of peritoneal macrophages were
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taken as parameters to evaluate the potential immunomodulatory activity of WPMPs in vitro, because splenocytes and macrophages are the most important immunocytes of immunity system, the splenocytes proliferation is crucial to cellular and humoral
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immune responses.[30]
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immune responses, and activated macrophages are essential to the innate and adaptive
Activation effect of WPMPs on splenocytes and peritoneal macrophages in vitro.
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As shown in figure 5a, WPMPs increased the cell viability of splenocytes in a dose-dependent manner at concentration from 20 to 500 µg/mL compared to the
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normal control group (##p < 0.05). It was illustrated that all the WPMPs samples could promote the proliferation of splenocytes. Comparing the promotion effect of WPMP, WPMP-1 and WPMP-2, WPMP-2 showed the strongest active effect on proliferation of splenocytes, followed by WPMP and WPMP-1. As shown in figure 5b, WPMPs could enhance the phagocytic capacity of peritoneal macrophages. Compared to the control group, 20 µg/mL WPMP and WPMP-2 could significantly enhanced the uptake of neutral red (p < 0.05), while that of WPMP-1 was 100µg/mL, and the enhancement was stronger with the increasing of concentration of polysaccharides. What’s more, the effect of WPMP-2 (500 µg/mL)
ACCEPTED MANUSCRIPT was higher than that of LPS group (p < 0.01). The result indicated that the acidic purified polysaccharides WPMP-2 demonstrated much stronger activation effect on
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phagocytosis of peritoneal macrophages than WPMP-1.
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Figure 5. Effects of different concentrations of WPMPs on(a) proliferation of splenocytes, the cell viability of untreated splenocyte was calculated as 100%, (b) Phagocytosis of peritoneal macrophages. The phagocytosis index of untreated peritoneal macrophages was calculated as 100%. The groups with RPMI 1640 medium and LPS (10 μg/mL) were the normal and model control group, respectively. The result repre-sented the mean ± SD (n = 3), #p < 0.05 and
##
p<
0.01 compared with the normal control group; ★p < 0.05 and ★★p < 0.01 compared with the LPS group.
ACCEPTED MANUSCRIPT Cytoprotective effect of WPMPs on 5-Fu treated splenocytes and peritoneal macrophages. 5-Fu is a representative anticancer agent but with severe side effects such as immunosuppression, etc.[7,31] As shown in figure 6, the proliferation and phagocytosis of splenocytes/peritoneal macrophages treated with 5-Fu (20 μg/mL) were markedly decreased compared with the normal control group (p < 0.01).
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Conversely, the proliferation of splenocytes and phagocytosis of peritoneal macrophage treated with mixture polysaccharides and 5-Fu were all significantly
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recovered compared with these of splenocytes/peritoneal macrophage treated with 5-Fu alone (p < 0.05), and the degree of recovery increased with the dose of
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polysaccharides increased. It was indicated that WPMPs could protect immunocytes aganist 5-Fu induced cellular damage and inhibition. And WPMP-2 demonstrated
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much stronger protective effect on splenocytes/peritoneal macrophages than WPMP
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and WPMP-1.
ACCEPTED MANUSCRIPT Figure 6. Cytoprotective effects of WPMPs on 5-Fu treated (a) splenocytes, the cell viability of normal group was calculated as 100%, (b)peritoneal macrophages. Phagocytosis index of normal group was calculated as 100%. The groups with RPMI 1640 medium and 5-Fu (20μg/mL) were the normal and model control group, respectively. The result represented the mean ± SD (n = 3), p < 0.01 compared with the normal control group; ★p < 0.05 and ★★p < 0.01 compared with the
##
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model control group.
Effects of WPMPs on IL-2 and TNF-α secretion. To further study the
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immunomodulatory activity of WPMPs, IL-2 and TNF-α levels in the supernatants of
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cell cultures of 5-Fu treated splenocytes and macrophages in vitro were measured by ELISA, because cytokines (IL-2, TNF-α, etc.) are signaling molecules that control
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homeostasis of the organism by regulating cell differentiation, proliferation, and apoptosis, as well as defense functions.[17, 32-35].
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As shown in figure 7, 5-Fu demonstrated strong inhibiting effect on IL-2 (962.6±37.3ng/L) and TNF-α (637.9±24.5ng/L) secretion compared with the normal control group (1175.3±34.6ng/L and 776.1±35.9ng/L, respectivly) (p < 0.01).
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However, there was a dose-dependent upregulation of IL-2 secretion in the groups of
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combinative administration of 5-Fu and WPMPs (figure 7a). What’s more, WPMPs showed significantly activation effect on the expression of IL-2 (≥1257.9±64.2 ng/L)
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at high concentrations (500 μg/mL), which was visible higher than that of normal control group (p < 0.01). The similar increasing of TNF-α secretion in the supernatant
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of macrophage cultures after combined administration of 5-Fu and WPMPs was also observed (figure 7b). Particularly, the TNF-α level of WPMP-2 (≥100 μg/mL) was significantly higher compared to normal control group (p < 0.01),and WPMP-2 showed much more excellent TNF-α secretion upregulate activity than the others. It was indicated that WPMPs could play a role of immunomodulator by stimulate the secretion of cytokines.
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ACCEPTED MANUSCRIPT
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Figure 7. Effects of the WPMPs on splenocytes IL-2 (a) and macrophages TNF-α (b) production.
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RPMI 1640 medium and 5-Fu (20 μg/mL) were the normal and model control group, respectively. The result represented the mean ± SD (n = 3). ##p < 0.01 compared with the normal control group; p < 0.05 and ★★p < 0.01 compared with the model control group.
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★
3.4 Structure-activity relationship of WPMPs
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It is well known that the molecular weights, monosaccharide composition, functional groups, branching degrees, and glycosidic linkages were some of the structural features identified as determining or contributing to the polysaccharides immunostimulatory activity.[36] Generally, a high branching degree is benefit to the formation of triple helices in D-glucans which is positively associated to immunostimulatory activity.[37,38] The degree of branching and uronic acids have been regarded as important roles in antioxidant activity,[24,39,40] and many acid polysaccharides were proved to be active in antitumor and immunomodulatory.[27,39,40] The presence of acetyl and sulfate groups of mannans, galactans, and fucoidans are
ACCEPTED MANUSCRIPT crucial structural features for immunostimulatory activity.[39,40] In some cases, molecular weight could be not as relevant as other structural features,[36] etc. However, these structural features may have different relevance depending on the type of polysaccharides and their combination may impact the resulting activity in different ways.
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The potential immunomodulatory activity of WPMPs could be comprehensively evaluated by combining the activation and cytoprotection activity on immunocytes. It
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was indicated that the immunomodulatory activity of WPMP-2 was markedly better than that of WPMP-1 and WPMP. Since WPMP was the mixture of WPMP-1and
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WPMP-2, the bioactivity of WPMP could be regarded as the aggregated results of WPMP-1and WPMP-2. Considering the molecular weight, content of uronic acid, and
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degree of branching of WPMP-2 were all higer than those of WPMP-1, and according to the above literatures on structure-activity relationship of polysaccharides, the
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presence of uronic acid group, and higher branching degree and molecular weight were suggested to be the positive characteristics for the better immunostimulatory
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4. Conclusions
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activity.
In this study, two completely different homogeneous polysaccharides named
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WPMP-1 and WPMP-2 were purified from Polygonum multiflorum and their structural features and immunomodulatory activity have been investigated. The
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molecular weight of two homogeneous polysaccharides were 2.04kDa and 92.13kDa, respectively. WPMP-1 was constituted by 1,4-linked α-D-Glcp as backbone, with a side branch at O-6 position of 1,4,6-linked α-D-Glcp. WPMP-2 was constituted by 1,2-linked α-D-Rhap, 1,2,4-linked α-D-Rhap, and 1,4-linked α-D-GalpA as the backbone, with side chain of either arabinan (1,3,5-linked α-L-Araf, 1,5-linked α-L-Araf, terminal α-L-Araf) or galactooligosaccharides (1,4-linked β-D-Galp, 4-linked
α-D-GalpA)
Immunomodulation
on
assays
O-2/4
position
of
indicated
that
WPMPs
1,2,4-linked
α-D-Rhap.
possessed
significant
immunomodulatory activity by promoting proliferation of splenocytes and activating
ACCEPTED MANUSCRIPT phagocytosis of peritoneal macrophages, and also protecting immunocytes against 5-Fu induced immunosuppression. Moreover, due to the presence of uronic acid group, and higher branching degree and molecular weight, WPMP-2 doing much better both in activation and cytoprotection than WPMP-1. It was illustrated that polysaccharides presented in Polygonum multiflorum were definitely worth further
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exploring, and utilized as immunomodulator contribute to food and pharmaceutical industries, particularly, WPMP-2 could be more efficacious in lessening
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chemotherapy induced immunosuppression.
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Acknowledgements
This research work was supported by Innovative Project on Designing and Screening
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Drug Candidates of Chongqing (cstc2015zdcy-ztzx120003), the Key Project of Frontier and Basic Research of Chongqing Science and Technology Committee
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(cstc2015jcyjBX0010), and Innovation Project Program of Chongqing Science and Technology Committee (cstc2015shmszx00014).
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Notes
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The authors declare no competing financial interest.
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