Carbohydrate Polymers 127 (2015) 94–100

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Isolation, purification and structural characterization of polysaccharide from Acanthopanax brachypus Haobin Hu a,∗ , Haipeng Liang b , Yun Wu a a b

College of Chemistry & Chemical Engineering, Longdong University, Qingyang 745000, PR China Department of Oncology, Qingyang First People’s Hospital, Qingyang 745000, PR China

a r t i c l e

i n f o

Article history: Received 27 December 2014 Received in revised form 10 March 2015 Accepted 15 March 2015 Available online 28 March 2015 Keywords: Acanthopanax brachypus Polysaccharide Isolation Pruification Structural characterization

a b s t r a c t A water-soluble polysaccharide, designated as ABPS-21, was isolated from the stem barks of Acanthopanax brachypus by hot-water extraction and purified by Cellulose DEAE-52 and Sephacryl S-300 HR gel-filtration chromatography, respectively. The homogeneity and molecular weight were determined using HPGPC. The structure was elucidated based on monosaccharide composition and methylation analysis, partial acid hydrolysis, periodate oxidation, Smith degradation, IR and NMR spectroscopy. The result showed that ABPS-21 was a homogeneous heteropolysaccharide including galactose, glucose, rhamnose and galacturonic acid in a molar ratio of 3.0:2.0:2.0:1.0, respectively, with an average molecular weight of 1.06 × 105 Da. The main chain of ABPS-21 was made up of →4)-␤-d-Galp-(1→4)-␤-d-Glcp-(1→4)␤-d-Galp-(1→4)-␤-d-Galp-(1→2)-␣-l-Rhap-(1→, and two side chains ␣-l-Rhap-(1→4)-␤-d-Glcp-(1→ and ␣-d-galpA-(1→ were attached to the backbone chain at O-6 position of 1,4,6-linked ␤-d-Glcp and 1,4,6-linked ␤-d-Galp, respectively. ABPS-21, an acidic branched polysaccharide from A. brachypus, was not described up to now. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Polysaccharides, a class of polymeric carbohydrates consisting of number of monosaccharides linked by glycosidic bonds in branched or unbranched chains (Zong, Cao, & Wang, 2012), have many biological functions like energy storage, structure support, anti-inflammation, antioxidation, antitumor, and so on (Hu, Cheong, Zhao, & Li, 2013; Jiang et al., 2013; Huang, Tan, Tan, & Peng, 2011). Because the separation, purification, composition determination, and structural analysis of polysaccharides have made remarkable progress, as well as because the biological functions of polysaccharides are further understood, polysaccharides have become one of research hotspots of natural medicines in recent years. Acanthopanax species (Araliaceae) are widely distributed throughout Korea, Japan, China, and the far-eastern regions of Russia (Phuong et al., 2006), and used in tradtional medicines in Eastern cultures. In vitro and in vivo experimental studies have demonstrated that many Acanthopanax species exhibit multiple biological effects against oxidative stress, diabetes, inflammation, cancer, and obesity (Jung, Lee, & Lee, 2012). Acanthopanax brachypus is

distributed in a narrow geographical area, most in the loess plateau of the northwest of China (Delectis Florae Reipublicae Popularis Sinicae, 1980). As a peculiar folk medicinal plant, A. brachypus is being developed in recent years due to containing many beneficial compounds, such as amino-acids, polysaccharides, organic acids, steroidal and triterpenoid saponins, and so on (Hu & Fan, 2012). However, to date, polysaccharides from A. brachypus have been seldom reported, even though polysaccharides as antioxidant, antitumor, antidiabetic and immunomodulatory substances, from Acanthopanax senticosus (Fu et al., 2012; Han et al., 2003), Acanthopanax obovatus (Wang, Tsumura, Ma, Shimura, & Ito, 1993), Acanthopanax giraldii (Wang, Tsumura, Shimura, & Ito, 1992), and Acanthopanax sessiliflorus (Lee et al., 2003) were well reported. In this study, a new water-soluble polysaccharide from A. brachypus was isolated and purified, then the structure was characterized. This will offer a theoretical basis for the further investigation on the structure-activity relationship and development of A. brachypus polysaccharides. 2. Experimental 2.1. Plant material

∗ Corresponding author. Tel.: +86 934 8522487; fax: +86 934 8651531. E-mail address: [email protected] (H. Hu). http://dx.doi.org/10.1016/j.carbpol.2015.03.066 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

The stem barks of A. brachypus was collected in October of 2010 from the Ziwuling mountains, (Gansu Province, China), and

H. Hu et al. / Carbohydrate Polymers 127 (2015) 94–100

identified by Prof. Guo Xiaoqiang working at College of Life-Science and Technology of Longdong University. A voucher specimen (No. 201010732) has been deposited in the Herbarium of College of LifeScience and Technology, Longdong University, Qingyang 745000, PR China. The stem barks were washed and dried in the shade. After that, the dried plants were powdered by using grinder. 2.2. Chemicals and instrumentations UV spectra was obtained on a Shimadzu UV-2401 spectrometer (Shimadzu, Japan). IR spectra was recorded with KBr pellets on a Shimadzu 8400S FT-IR infrared spectrometer (Shimadzu, Japan). NMR spectra were measured on a Bruker AMX-500 (Bruker, Switzerland) with tetra-methylsilane (TMS) as an internal standard. GC–MS analysis was carried out on an Agilent GC 7890N/MSD 5973N (Agilent, America) with an DB-1701 (14% cyanide propyl-phenyl) methyl polysiloxane capillary column (30 m × 0.25 mm, film thinkness 0.25 mm). HPGPC was carried out with a TSK-GEL G4000SW column (7.8 mm × 300 mm) on an Agilent 1100 series system equipped with an Agilent RID10A refractive index detector. Cellulose DEAE-52 and Sephacryl S-300 HR were purchased from Hengxin Chemical Reagent Co., (Shanghai, China). All standard monosaccharides (d-galactose, l-rhamnose, l-arabinose, d-glucose, d-xylose, d-mannose and dgalacturonic acid), dextran series with varying molecular weights of 5000, 12,000, 25,000, 50,000, 80,000, 150,000, 270,000 and 410,000 Da were purchased from Shanghai Yuanju Bioscience Technology Limited Company (China). Trifluoroacetic acid (TFA) and 1-phenyl-3-methyl-5-pyrazolone (PMP) were purchased from Sinopharm Chemical Reagent (Shanghai, China). All of other chemicals and reagents in this study were analytical grade from Xi’an Chemical Co. (Xi’an, China), and water were distilled water. 2.3. General analytical methods The physical characteristics were analyzed by color and texture observation (Ge, Duan, Fang, Zhang, & Wang, 2009). The carbohydrate content of the polysaccharide was determined by the phenol-sulfuric acid colorimetric methods, using d-glucose as the standard (Huang et al., 2011). Gel permeation and anion exchange chromatography were monitored by assaying the total sugar content. The content of uronic acid was measured by photometry with m-hydroxydiphenyl at 525 nm using d-galacturonic acid as the standard (Gao et al., 2015). d- or l-Configurations of sugars were determined using the Gerwig method (Zhang, Liu, & Lin, 2014). 2.4. Isolation and purification of polysaccharide The air-dried powder of A. brachypus (500 g) was extracted with 95% ethanol (1 L) 40 ◦ C in a water bath for two times (each for 2 h) to remove the pigment, fat and inactivate enzyme, and then filtered and dried at 60 ◦ C. Subsequently, the dried residue were extracted three times (each time for 10 h) with distilled water at a ratio of 33.3 g/L (residue:distilled water) at 80 ◦ C. After centrifugation, the supernatant was combined and concentrated to its 1/4 total volume under reduced pressure in a rotary evaporator. The concentrated solution was deproteinated by the Sevag method (Yang, Huang, Wang, Cao, & Sun, 2008) for repeated 6 times. The solution was dialyzed against distilled water for 2 d, then was precipitated with EtOH up to 50%, and kept at 4 ◦ C overnight. After that, the precipitate was collected by centrifugation, dried in vacuo at room temperature, and washed successively with absolute ethanol, acetone and diethyl ether. Finally, the precipitate was dissolved in distilled water and then lyophilized in vacuum freeze dryer to

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obtain the crude polysaccharide (ABPS, 8.50 g). The protein content was determined by the Bardford’s method (Bradford, 1976) using ovalbumin as a standard. The decolorization of polysaccharide was measured using UV-spectrophotometry at 420 nm (Xie, Shen, Nie, Li, & Xie, 2011). ABPS sample (100 mg) was redissolved in distilled water, centrifuged, and purified with a Cellulose DEAE-52 column (2.6 cm × 40 cm) equilibrated with distilled water. The polysaccharides were fractionated eluting stepwise with distilled water, and followed by a NaCl gradient (0–1.2 M) at a flow rate of 1.0 mL/min. Each fraction (5 mL/tube) was checked at 490 nm by the phenolsulphuric acid method (Cuesta, Suarez, Bessio, Ferreira, & Massaldi, 2003). The eluted solution was separated into two fractions (ABPS-1 and ABPS-2), then major fraction ABPS-2 was further purified on Sephacryl S-300 HR gel-filtration column (1.6 cm × 80 cm) using distilled water as eluent at a flow rate of 15 mL/h. The elutes was concentrated, dialyzed against water, and finally lyophilized to obtain white fluffy pure polysaccharide ABPS-21 (41.9 mg). 2.5. Determination of homogeneity and relative molecular weight The homogeneity and molecular weight of ABPS-21 were identified by high-performance gel-permeation chromatography (HPGPC) (Cai, Xie, Chen, & Zhang, 2013). The HPGPC instrument was equipped with an Agilent 1100 HPLC system matched with a TSKGEL G4000SW column (7.8 mm × 300 mm) and an Agilent RID-10A refractive index detector. The sample was eluted with 0.7% Na2 SO4 at a flow rate of 0.6 mL/min. Data was analyzed by Agilent GPC Software. According to the peak shape of the HPGPC chromatogram, its homogeneity can be judged. A standard curve of the logarithm of relative molecular weight (MW ) related to retention time (t) was established using the dextrans of known molecular weights (5, 12, 25, 50, 80, 150, 270 and 410 kDa), and further calculated the relative molecular weight of ABPS-21 based on calibration curve (log MW = 9.08423–0.19415t, R2 = 0.9879). 2.6. Monosaccharide composition analysis of ABPS-21 The composition analysis of polysaccharide is an important step to control the quality and got basic information about the polysaccharide. The monosaccharide composition of ABPS-21 was determined by the PMP-labeling procedure (Li, Pan, Xia, Zhang, & Wu, 2014). Briefly, 5 mg ABPS-21 was hydrolyzed with 2 M TFA (trifluoroacetic acid, 3 mL) at 120 ◦ C for 6 h in a sealed glass tube. After completing the hydrolysis, the solution was concentrated to dryness (