Structural, physicochemical, antioxidant and antitumor property of an acidic polysaccharide from Polygonum multiflorum

International Journal of Biological Macromolecules 96 (2017) 494–500

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Structural, physicochemical, antioxidant and antitumor property of an acidic polysaccharide from Polygonum multiflorum Weili Zhu a , Xiaoping Xue a,∗ , Zhanjun Zhang b a b

Department of Blood Transfusion, Subei People’s Hospital of Jiangsu Province, Yangzhou 225001, Jiangsu, China College of Biological and Chemical Engineering, Yangzhou Vocational University, Yangzhou 225009, Jiangsu, China

a r t i c l e

i n f o

Article history: Received 14 September 2016 Received in revised form 12 December 2016 Accepted 18 December 2016 Available online 26 December 2016 Keywords: Acidic polysaccharide Bioactivities Physicochemical property Polygonum multiflorum Structural characterization

a b s t r a c t In this study, the structural characterization, physicochemical property, antioxidant and antitumor activity of an acidic polysaccharide (APS) from Polygonum multiflorum were investigated. Monosaccharide composition analysis showed APS was composed of arabinose, rhamnose, galactose and galacturonic acid in the molar ratio of 1.23:1.32:1.48:1.00. The presence of uronic acid was also confirmed by the bands at 1740, 1645 and 1425 cm−1 on Fourier transform-infrared spectroscopy. Methylation and nuclear magnetic resonance analyses showed APS was mainly composed by the residues of →5)-␣-lAraf-(1→, →3)-␤-d-Galp-(1→, →3,6)-␤-d-Galp-(1→, →4)-␣-d-GalAp-(1→ and →2)-␣-l-Rhap-(1→ in the backbone. The non-reducing terminal ␣-l-Araf-(1→ was probably attached to the O-6 position of →3,6)-␤-d-Galp-(1→ residues. Besides, APS exhibited rod-like and flaky shapes with rough surface. The initial decomposition of APS occurred at 172 ◦ C, and the rapidest weight loss rate of APS appeared at 320 ◦ C. Antioxidant activity assay showed the DPPH radical scavenging activity of APS was 67.5% at 1 mg/mL. At the concentration of 400 ␮g/mL, the antiproliferation activities of APS against HepG-2 and BGC-823 cells were 65.28% and 51.57%, respectively. Our results suggested APS could be a potential antioxidant and antitumor agent. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The dried root of Polygonum multiflorum Thunb., well known as Heshouwu in China, is a popular traditional Chinese medicine [1]. The raw material of Heshouwu is commonly processed into Polygoni multiflori Radix Praeparata (also called Zhi-heshouwu in China) by specialized heating [2]. Till now, more than 100 kinds of chemical compounds have been successfully isolated from Heshouwu. Stilbene glycosides, anthraquinones, phenolics, phospholipids and carbohydrate compounds constitute the major components of Heshouwu [3]. Clinical studies have demonstrated that both crude and purified extracts from Heshouwu possess several bioactive properties, such as anti-aging, anti-hyperlipidaemia, anti-cancer, anti-inflammatory, immunomodulation and neuroprotective effects [4,5]. As important bioactive ingredients, polysaccharides have been isolated from both P. multiflorum and P. multiflori in recent years [6–9]. Lv et al. suggested that polysaccharides from P. multiflorum possessed strong antioxidant capacity against free radical, lipid

∗ Corresponding author. E-mail address: [email protected] (X. Xue). http://dx.doi.org/10.1016/j.ijbiomac.2016.12.064 0141-8130/© 2016 Elsevier B.V. All rights reserved.

oxidation and protein glycation [6]. Other researchers reported that polysaccharides from P. multiflori had antioxidant, anti-anemia and immunomodulation activities [7,8]. However, the detailed structures and other biological activities of polysaccharides from P. multiflorum and P. multiflori are still unknown. In our previous study, polysaccharides were extracted from the roots of P. multiflorum by the ultrasonic-assisted method. Two polysaccharide fractions (neutral and acidic polysaccharides) were obtained through purification on DEAE-cellulose column. The main neutral polysaccharide fraction was characterized as a linear (1 → 6)-␣-dglucan with potential antiproliferation activity against HepG-2 and BGC-823 tumor cells [9]. In general, acidic polysaccharides possess more pronounced bioactivities than neutral polysaccharides of the same origin [10]. Therefore, we investigated the structural characterization and biological activities of the acidic polysaccharide from P. multiflorum in this study. The structural characterization of polysaccharide was characterized by gas chromatography (GC), high performance size exclusion chromatography (HPSEC), Fourier transform-infrared (FT-IR), methylation analysis and nuclear magnetic resonance (NMR). Afterwards, the physicochemical properties of polysaccharide including surface morphology, thermal stability and crystallinity were determined by scanning electron microscopy

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(SEM), thermal gravimetric analysis (TGA) and X-ray diffraction (XRD). Finally, the antioxidant and antitumor activities of polysaccharide were evaluated. 2. Materials and methods 2.1. Reagents and cell lines Deuterium oxide (D2 O) and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) were purchased from Sigma Chemical Co. (MO, USA). HepG 2 human hepatocellular carcinoma cell line and BGC-823 human gastric carcinoma cell line were supplied by the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) supplemented with 100 U/mL of penicillin and 100 ␮g/mL of streptomycin (Gibco BRL, NY, USA), and were incubated at 37 ◦ C under a humidified atmosphere containing 5% CO2 . 2.2. Extraction and purification of polysaccharides from P. multiflorum Crude polysaccharides were extracted from roots of P. multiflorum (Xinyang Medicine Co., Henan, China) by the ultrasoundassisted extraction (UAE) method according to our previous report [9]. Under the optimal UAE conditions (ultrasonic power 158 W, extraction temperature 62 ◦ C, extraction time 80 min, and ratio of water to material 20 mL/g), the maximum extraction yield of crude polysaccharides was 5.49%. The obtained crude polysaccharides were then purified on DEAE-52 anion exchange column (2.6 × 30 cm) to afford a neutral polysaccharide fraction (named as F-1) and an acidic polysaccharide fraction (named as F-2) [9]. The acidic polysaccharide fraction (F-2) was further purified on Sepharose CL–4 B column (1.6 × 80 cm). The resultant main polysaccharide fraction (named as APS) was dialyzed against distilled water and freeze-dried for further study.

495

with a 4 cm−1 resolution. For FT-IR measurement, 2 mg of APS sample was ground with potassium bromide (KBr) powder and pressed into pellet. 2.3.4. Methylation and GC–MS analysis Before methylation, APS was carboxy-reduced by the carbodiimide method [13]. Then, both native and carboxyreduced APS were methylated according to the reported method [14]. The completely methylated APS was confirmed by FT-IR and further subjected to hydrolyzation by trifluoroacetic acid, reduction with NaBH4 and acetylation by acetic anhydride. The resultant partially methylated alditol acetate derivatives were identified by their retention times and electron ionization spectra on Varian CP-3800 GC coupled with Saturn 2000 ion trap mass spectrometer (Walnut Creek, USA) and DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 ␮m, J&W Scientific, USA). 2.3.5. NMR analysis For NMR analysis, freeze-dried APS was kept over P2 O5 under vacuum for several days and then dissolved in D2 O. The 1 H and 13 C NMR spectra of APS were recorded on AVANCE-600 spectrometer (Bruker Inc., Germany) at 25 ◦ C. The instrument was operated at 600 MH and 150 MHz for 1 H NMR and 13 C NMR, respectively. 2.4. Physicochemical properties of APS The surface morphology of APS was observed on S-4800 SEM (Hitachi Ltd., Japan) at an accelerating voltage of 10 kV with a working distance of 7.1 mm. APS sample was sputter-coated with gold layer before observation. Thermal gravimetric analysis of APS was carried out on Pyris 1 TGA (PerkinElmer Inc., USA) under nitrogen flow by heating from 30 to 800 ◦ C with heating rate of 10 ◦ C/min. The powder XRD measurement of APS was recorded on D8 Advance X-ray diffractometer (Bruker AXS, Germany) using Cu K␣ radiation ( = 1.5406 Å) with scattering angles (2) ranging from 10◦ to 40◦ .

2.3. Structural characterization of APS 2.5. Antioxidant activity of APS 2.3.1. Monosaccharide composition analysis The monosaccharide composition of APS was analyzed by the reported method [11]. Briefly, APS was first subjected to acidic hydrolysis in 2 M trifluoroacetic acid at 120 ◦ C for 2 h, and followed by conversion of hydrolyzate into trimethylsilyl derivatives. The trimethylsilyl derivatives were analyzed on Agilent 6890A GC system (Agilent Technologies, USA) equipped with flame-ionization detector. A HP-5 fused-silica capillary column (30 m × 0.25 mm × 0.25 ␮m) was used to separate and quantify monosaccharide composition of APS. The uronic acid content in APS was measured by m-hydroxydiphenyl colorimetric method using d-glucuronic acid as the standard [12]. 2.3.2. Determination of the molecular weight The molecular weight of APS was determined by HPSEC on Agilent 1100 system (Agilent Technologies, CA, USA) equipped with evaporative light scattering detector. Based on our previously established method [9], a TSK gel G4000 PWXL column (30 cm × 7.8 mm × 10 ␮m, Tosoh Corp., Tokyo, Japan) column was eluted with distilled water at 50 ◦ C and flow rate of 0.6 mL/min. The molecular weight of APS was calculated according to the calibrated curve established by standard dextran T-series (T-500, T-200, T100, T-50 and T-10). 2.3.3. FT-IR analysis The FT-IR spectroscopy of APS was recorded on Tensor-27 spectrometer (Bruker Optics, France) over the range of 400–4000 cm−1

The antioxidant activity of APS was evaluated by DPPH radical scavenging activity according to reported method [15]. Briefly, 0.2 mL of DPPH solution (0.4 mM DPPH in methanol) was mixed with 1.0 mL of sample (0.1–1 mg/mL) and 2.8 mL of distilled water. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min under darkness. DPPH radical scavenging activity of APS was calculated by measuring the absorbance of reaction mixture at 517 nm. 2.6. Antitumor activity of APS The antitumor activity of APS against human hepatoma HepG-2 cells and gastric cancer BGC-823 cells was determined as reported previously [9]. Briefly, sterilized APS solution (50, 100, 200 and 400 ␮g/mL in fresh medium, 50 ␮L/well) and tumor cells (2 × 105 cells/mL, 100 ␮L) were placed on a 96-well plate and cultured for 48 h. The inhibitory ratio of APS against tumor cell proliferation was calculated by measuring cell viability using MTT assay. 2.7. Statistical analysis Data were expressed as mean ± standard deviation (SD) of triplicates. Statistical analysis was performed using SPSS software of version 13.0 (Chicago, IL, USA). Difference was considered to be statistically significant if p < 0.05.

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3. Results and discussion 3.1. Isolation and purification of acidic polysaccharide In our previous work, crude polysaccharides were isolated from the dried roots of P. multiflorum by UAE method. The obtained crude polysaccharides were purified on DEAE-52 anion exchange chromatography to afford a neutral polysaccharide and an acidic polysaccharide [9]. In this study, the acidic polysaccharide fraction was further purified on Sepharose CL–4 B gel filtration chromatography to yield only one single and symmetrical elution peak (named as APS) (Fig. 1a). This indicated that APS was a homogeneous polysaccharide. The recovery ratio of APS was 18.4% based on crude polysaccharides. The molecular weight of APS was measured by HPSEC. As shown in Fig. 1b, APS only showed a symmetrical peak on HPSEC, further indicating APS was a homogeneous polysaccharide. Based on the calibration curve established by standard dextran T-series, the molecular weight of APS was determined as 1.15 × 106 Da. The molecular weight of APS was higher than hot water extracted polysaccharides from P. multiflorum (4.8 × 105 and 6.1 × 105 Da), the alkali extracted polysaccharide from P. multiflori (7725 Da) and ultrasound-assisted extracted neutral polysaccharide from P. multiflori (3.26 × 105 Da) [6,8,9]. 3.2. Monosaccharide composition of APS GC analysis showed APS was composed of arabinose, rhamnose and galactose in the molar ratio of 1.00:1.07:1.20 (Fig. 2). Apart from neutral sugars, the uronic acid content in APS was determined as 22.5% by m-hydroxydiphenyl colorimetric method. Thus, APS was composed of arabinose, rhamnose, galactose and uronic acid in the molar ratio of 1.23:1.32:1.48:1.00. The monosaccharide composition of APS was significantly different from that of other polysaccharides from P. multiflorum. Lv et al. found that both neutral and acidic polysaccharide fractions from water extract of P. multiflorum were composed of glucose [6]. Chen et al. reported that polysaccharide from P. Multiflori was composed of rhamnose, arabinose, xylose and glucose in the molar ratio of 1.64:1.00:1.34:6.06 [7]. Zhang et al. suggested that the alkali-extracted polysaccharide from P. multiflori was composed of galactose, xylose, arabinose, mannose, galacturonic acid and glucose [8]. In our previous work, the neutral polysaccharide isolated from P. multiflorum was mainly composed of glucose [9]. 3.3. FT-IR spectroscopy of APS The FT-IR spectroscopy of APS was presented in Fig. 3. The strong band at around 3400 cm−1 was assigned to hydroxyl stretching vibration. The characteristic band of polysaccharide appeared at 2940 cm−1 was attributed to C H asymmetric vibration. The band at 1740 cm−1 was due to the vibration of esterified carboxyl group, while the bands at 1645 and 1425 cm−1 were attributed to the vibration free carboxyl group [16]. In addition, several bands ranging from 1150 to 1010 cm−1 was assigned to C O stretching vibrations [7]. 3.4. Methylation analysis of APS Methylation coupled with GC–MS analysis was used to deduce the glycosidic linkages of APS. In order to identify uronic acid residue and its linkage pattern, both native APS and its carboxylreduced product (APS-R) were methylated. The fully methylated products of APS and APS-R were hydrolyzed, and then converted into alditol acetates followed by GC–MS analysis. As shown in Table 1, the sole terminal residue was T-Araf (16.23%). Araf residues also existed in the 1,5-linked form (7.76%). The presence of 1,2-

Table 1 Partial methylated alditol acetates and deduced glycosidic linkages of native and carboxy-reduced APS. Partially methylated alditol acetates

b

2,3,5-Me3 -Ara 2,3-Me2 -Ara 3,4-Me2 -Rha 2,4,6-Me3 -Gal 2,3,6-Me3 -Gal 2,4-Me2 -Gal a b c

Deduced glycosidic linkages

T-Araf-(1→ →5)-Araf-(1→ →2)-Rhap(1→ →3)-Galp-(1→ →4)-Galp-(1→ →3,6)-Galp-(1→

Relative molar ratio (%) APS

APS-Ra

20.75 10.01 32.63 14.32 n.d.c 22.29

16.23 7.76 24.33 10.53 24.54 16.61

APS-R, carboxy-reduced APS. 2,3,5-Me3 -Ara, 2,3,5-tri-O-methyl-1,4-di-O-acetyl-arabinitol, etc. n.d., not detected.

linked Rhap residues (24.33%) was also detected. The Galp residues were mainly 1,3-linked (10.53%) and 1,3,6-linked (16.61%), indicating APS was a branched polysaccharide. By comparing with methylated APS, methylated APS-R had higher content of 2,3,6Me3 -Galp (24.54%). This indicated that the 2,3,6-Me3 -Galp in APS-R resulted from the carboxyl-reduced 1,4-linked GalpA, which was also found in other polysaccharides [17,18]. Notably, the relative mole ratio of T-Araf was approximately equal to that of →3,6)Galp-(1→, indicating the non-reducing terminal Araf was probably attached to the O-6 position of Galp residues. In addition, the mole ratio of →2)-Rhap-(1→ was somewhat equal to that of →4)GalAp-(1→, suggesting these two residues probably appeared as disaccharide units of →4)-GalAp-(1 → 2)-Rhap-(1→. 3.5. NMR analysis of APS The glycosidic linkage patterns were further confirmed by NMR technique. 1 H NMR spectroscopy of APS was shown in Fig. 4a. Based on the results of methylation analysis, different anomeric proton signals could be assigned. The overlapped anomeric proton signal at chemical shift of 5.20 ppm was assigned to the H-1 of →2)-␣-l-Rhap-(1→ (residue F) and ␣-l-Araf-(1→ (residue A) [17,18]. The anomeric proton signals at 5.05 and 4.96 ppm were attributed to H-1 of →5)-␣-l-Araf-(1→ (residue B) and →4)-␣d-GalAp-(1→ (residue E), respectively [19]. Two anomeric proton signals appeared at 4.47 and 4.37 ppm could be assigned to H1 of →3,6)-␤-d-Galp-(1→ (residue D) and →3)-␤-d-Galp-(1→ (residue C), respectively [17,19]. In addition, the signal at 1.24 ppm should be due to −CH3 group of →2)-␣-l-Rhap-(1→ (residue F) [19]. As shown in Fig. 4b, the 13 C NMR spectrum of APS exhibited several anomeric carbon signals ranging from 97.7 to 109.3 ppm. Signals at 109.3 and 107.4 ppm were assigned to C-1 of ␣-l-Araf(1→ (residue A) and →5)-␣-l-Araf-(1→ (residue B), respectively [20,21]. Two anomeric carbon signals at 104.3 and 103.7 ppm were attributed to the C-1 of →3)-␤-d-Galp-(1→ (residue C) and →3,6)-␤-d-Galp-(1→ (residue D), respectively [10,22]. The signals of disaccharide units appeared at 98.9 and 97.7 ppm were assigned to the C-1 of →4)-␣-d-GalAp-(1→ (residue E) and →2)-␣-l-Rhap(1→ (residue F), respectively [23]. The C-6 signals of residues E and F appeared at 176.4 and 16.5 ppm, respectively. Other carbon chemical shifts were assigned according to literature and listed in Table 2. Based on the results of methylation analysis and NMR spectra of APS, the structure of APS was predicted and shown in Fig. 4c. 3.6. Physicochemical property of APS The surface morphology of APS was observed by SEM. As shown in Fig. 5, APS showed both rod-like and flaky shapes. The surface of APS was also much rough. Notably, some tiny pores existed in the centre of flaky APS. These tiny pores probably formed during the

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(a)

497

Absorbence (490 nm)

1.5

APS

1.2 0.9 0.6 0.3 0 0

10

20

30

40

50

Tubes

(b) Signal Voltage (mV)

250 200 150 100 50 0 0

5

15

10 Time (min)

20

Fig. 1. Elution profile of the acidic polysaccharide fraction on Sepharose CL–4 B gel filtration column (a) and HPSEC (b).

(a) pA D-Glucose D-Mannose

1200

D-Glactose

D-Fructose

D-Xylose

L-Fucose

1600

L-Rhamnose

2000

L-Arabinose

Erythitol

2400

800

400

16

18

20

22

24

26

28

24

26

28

30

min

30

min

(b)pA 600

300

L-Arabinose L-Rhamnose

Erythitol

400

D-Glactose

500

200

100

16

18

20

22

Fig. 2. GC chromatograms of trimethylsilyl derivatives of standard monosaccharide mixtrue (a) and APS hydrolyzate (b).

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Table 2 Chemical shifts of different glycosyl residues of APS in 13 C NMR spectroscopy. Glycosyl residues

C−1

C−2

C−3

C−4

C−5

C−6

A: ␣-l-Araf-(1→ B: →5)-␣-l-Araf-(1→ C: →3)-␤-d-Galp-(1→ D: →3,6)-␤-d-Galp-(1→ E: →4)-␣-d-GalAp-(1→ F: →2)-␣-l-Rhap-(1→

109.2 107.4 104.3 103.7 98.9 97.7

81.3 81.3 70.2 70.2 72.6 76.6

76.5 76.5 80.6 81.3 78.4 68.6

83.9 82.1 68.6 68.6 83.5 71.4

61.0 67.9 75.1 73.4 73.4 69.4

61.0 69.4 176.4 16.5

120

UAE process and could render APS good water binding ability. The thermal property of APS was measured by TGA technique. In TGA curve, the weight of APS was presented as a function of temperature. The first derivative thermogravimetric (DTG) curve supplied additional information on the relative decomposition rate of APS. As shown in Fig. 6, the depolymerization, decomposition and degradation of APS occurred when temperature reached 172 ◦ C. In addition, the rapidest weight loss rate of APS appeared at 320 ◦ C. The powder XRD profile of APS was presented in Fig. 7. A broad peak appeared at approximately 23◦ (2␪), which was due to the polymeric structure of APS. This suggested APS had an amorphous structure [24].

100 Transmittance (%)

3.7. Antioxidant and antitumor activities of APS 80 60

2940

1740 1425

40

640

1645 1090

20 3400 0 4000

3100

2200 −1 Wavenumber (cm )

1300

400

Fig. 3. FT-IR spectrum of APS in KBr pellet over the range of 400–4000 cm−1 .

DPPH radical scavenging assay is widely used to evaluate the antioxidant activity of compounds [25]. This assay is based on the reduction of DPPH radical into non-radical form of DPPH-H in the presence of hydrogen donating antioxidant. The positive correlation between APS concentration and DPPH radical scavenging activity was well presented in Fig. 8. At the concentration of 1 mg/mL, DPPH radical scavenging activities of APS and ascorbic acid (positive control) were 67.5% and 98.3%, respectively. In addition, the IC50 values for the DPPH scavenging activities of APS and ascorbic acid were 0.26 and 0.09 mg/mL, respectively. This indicated APS had strong in vitro antioxidant activity. Lv et al. also suggested that polysaccharides from P. multiflorum exerted antiox-

Fig. 4. 1 H NMR (a) and 13 C NMR (b) spectra of APS in D2 O with the predicted structure (c). Residues A, B, C, D, E and F represented ␣-l-Araf-(1→, →5)-␣-l-Araf-(1→, →3)-␤-d-Galp-(1→, →3,6)-␤-d-Galp-(1→, →4)-␣-d-GalAp-(1 → and →2)-␣-l-Rhap-(1→, respectively.

W. Zhu et al. / International Journal of Biological Macromolecules 96 (2017) 494–500

499

Fig. 7. XRD profile of APS with scattering angles (2) ranging from 10 to 40◦ .

Scavenging activity (%)

100 80 60 40

APS Ascorbic acid

20 0 0

0.2

0.4

0.6

0.8

1

Concentration (mg/mL) Fig. 8. DPPH radical scavenging activity of APS using ascorbic acid as the positive control. Data represents mean ± SD of triplicates.

100

0

80

-2

Weight (%)

-4 60 -6 40 -8 20

DTG (°C/min)

Fig. 5. SEM micrographs of APS at magnifications of 2000× and 7000× .

-10

0

-12 0

200

400

600

800

Temperature (°C) Fig. 6. TGA and DTG curves of APS by heating from 30 to 800 ◦ C.

Fig. 9. Antiproliferation activities of APS against human hepatoma HepG-2 cells and gastric cancer BGC-823 cells using 5-fluorouracil (5-FU, 50 ␮g/mL) as the positive control. Data represents mean ± SD of triplicates.

idant activity by scavenging free radicals (e.g., superoxide anion radical, hydroxyl radical, and hydroxyl peroxide), lipid oxidation and protein glycation [6]. Moreover, the intraperitoneal administration of polysaccharide from P. Multiflori could enhance the serum antioxidant profiles in cyclophosphamide-induced anemic mice [7]. These results indicated polysaccharides from P. multiflorum and P. Multiflori could be used as novel natural antioxidant agents. The antitumor activities of APS against human hepatoma HepG2 cells and gastric cancer BGC-823 cells were shown in Fig. 9. The antitumor activity was closely related to the concentration of APS. At the concentration of 400 ␮g/mL, the antitumor activi-

ties of APS against HepG-2 and BGC-823 cells reached 65.28% and 51.57%, respectively. The IC50 values for the antitumor activities of APS against HepG-2 and BGC-823 cells were 0.11 and 0.31 mg/mL, respectively. In our previous work, the antitumor activities of the neutral polysaccharide from P. multiflorum against HepG-2 and BGC-823 cells were 53.35% and 38.58%, respectively [9]. By contrast, APS showed higher antitumor activity in vitro than the neutral polysaccharide. The difference in the antitumor activity between neutral and acidic polysaccharides should be attributed to the different monosaccharide compositions, molecular weights, glycosidic linkages as well as uronic acid contents. Our results indicated APS could also be utilized as a novel antitumor agent.

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4. Conclusion In this study, an acidic polysaccharide was isolated from the root of Polygonum multiflorum. The structural, physicochemical, antioxidant and antitumor property of the acidic polysaccharide were investigated in detail. Structural characterization showed APS was mainly composed of the linear backbone containing →5)-␣-l-Araf(1→, →3)-␤-d-Galp-(1→, →3,6)-␤-d-Galp-(1→, →4)-␣-d-GalAp(1→ and →2)-␣-l-Rhap-(1→ residues with non-reducing terminal ␣-l-Araf-(1→ probably attached to the O-6 position of →3,6)-␤-dGalp-(1→ residues. Physicochemical study further indicated that the tiny pores observed by SEM could give APS good water binding ability. Moreover, APS exhibited good DPPH radical scavenging activity and antiproliferation activity against human hepatoma HepG-2 cells and gastric cancer BGC-823 cells. Our results suggested APS could be used as a potential natural antioxidant and antitumor agent. Acknowledgment This work was supported by the Scientific Research Foundation of Subei People’s Hospital of Jiangsu Province (yzucms201631). References [1] Z. Liu, Y. Liu, C. Wang, N. Guo, Z. Song, C. Wang, L. Xia, A. Lu, Comparative analyses of chromatographic fingerprints of the roots of Polygonum multiflorum Thunb. and their processed products using RRLC/DAD/ESI-MSn, Planta Med. 77 (2011) 1855–1860. [2] Y.H. Lo, Y.J. Chen, T.Y. Chung, N.H. Lin, W.Y. Chen, C.Y. Chen, M.R. Lee, C.C. Chou, J.T. Tzen, Emoghrelin a unique emodin derivative in Heshouwu, stimulates growth hormone secretion via activation of the ghrelin receptor, J. Ethnopharmacol. 159 (2015) 1–8. [3] N. Saewan, A. Jimtaisong, Natural products as photoprotection, J. Cosmet. Dermatol. 14 (2015) 47–63. [4] L. Lin, B. Ni, H. Lin, M. Zhang, X. Li, X. Yin, C. Qu, J. Ni, Traditional usages, botany, phytochemistry, pharmacology and toxicology of Polygonum multiflorum Thunb.: a review, J. Ethnopharmacol. 159 (2015) 158–183. [5] G.A. Bounda, Y.U. Feng, Review of clinical studies of Polygonum multiflorum Thunb. and its isolated bioactive compounds, Pharmacognosy Res. 7 (2015) 225–236. [6] L. Lv, Y. Cheng, T. Zheng, X. Li, R. Zhai, Purification, antioxidant activity and antiglycation of polysaccharides from Polygonum multiflorum Thunb, Carbohydr. Polym. 99 (2014) 765–773. [7] Q. Chen, S.Z. Zhang, H.Z. Ying, X.Y. Dai, X.X. Li, C.H. Yu, H.C. Ye, Chemical characterization and immunostimulatory effects of a polysaccharide from Polygoni Multiflori Radix Praeparata in cyclophosphamide-induced anemic mice, Carbohydr. Polym. 88 (2012) 1476–1482. [8] Q. Zhang, Y. Xu, S. Zou, X. Zhang, K. Cao, Q. Fan, Novel functional polysaccharides from Radix Polygoni Multiflori water extracted residue: preliminary characterization and immunomodulatory activity, Carbohydr. Polym. 137 (2016) 625–631.

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