Aqueous biphasic system based on low-molecular-weight polyethylene glycol for one-step separation of crude polysaccharides from Pericarpium granati using high-speed countercurrent chromatography

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Aqueous biphasic system based on low-molecular-weight polyethylene glycol for one-step separation of crude polysaccharides from Pericarpium granati using high-speed countercurrent chromatography Xin-Yu Zhou, Jing Zhang, Rui-Ping Xu, Xue Ma, Zhi-Qi Zhang ∗ Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China

a r t i c l e

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Article history: Received 12 July 2014 Accepted 5 August 2014 Available online xxx Keywords: Aqueous biphasic system Low-molecular-weight polyethylene glycol Phase diagram Polysaccharides High-speed countercurrent chromatography

a b s t r a c t The aqueous biphasic system (ABS) plays a key role in the separation of bioactive substances, and the establishment and application of a low-molecular-weight polyethylene glycol (PEG) ABS remains a challenge in high-speed countercurrent chromatography (HSCCC). In this work, an ABS of low-molecularweight PEG, namely PEG400–Na2 SO4 –H2 O (20%–16%–64%, w/w/w), was developed on the basis of the phase diagram, and the phase forming time and ratio, and applied to HSCCC for the separation of polysaccharides. The crude polysaccharide extracted from Pericarpium granati (PGP) was successfully separated and three purified polysaccharides were obtained: PGP-1, with an average molecular weight of 13,210 Da and composed of xylose (12.4%), ribose (10.1%), and glucose (77.5%); PGP-2, which is a homogeneous polysaccharide with an average molecular weight of 2584 Da and consists of mannose; and PGP-3, with an average molecular weight of 2459 Da and composed of ribose (51.4%), mannose (26.7%), and glucose (21.9%). This success shows that an ABS based on low-molecular-weight PEG could be applied to HSCCC separation technology. © 2014 Elsevier B.V. All rights reserved.

1. Introduction High-speed countercurrent chromatography (HSCCC), a type of liquid–liquid partition chromatography, is a cost-effective separation technique, which can eliminate the complications resulting from the solid separation matrix [1]. HSCCC separation depends on the target compound’s distribution pattern in the diphase solvent system. Therefore, selection of the appropriate diphase solvent system for the target compound(s) is the key step in the development of an HSCCC separation process [2]. The aqueous biphasic system (ABS) plays a vital role in the separation of bioactive substances, because the native conformation and biological activity of biomolecules in the aqueous solution is preserved [3]. For HSCCC, the PEG/salt ABS is the most popular and widely applied solvent system. PEG is non-toxic, inexpensive, and biodegradable [4], and is used for various biotechnical and biomedical applications in aqueous solutions [5], although it is a polar molecule. Hydrophobicity

∗ Corresponding author. Tel.: +86 29 81530792; fax: +86 29 81530792. E-mail address: [email protected] (Z.-Q. Zhang).

increases with the increase in the molecular weight of PEG, facilitating the formation of the ABS. Therefore, the ABS based on high-molecular-weight (1000 and above) PEG has been successfully used in the HSCCC separation of liquiritigenins [6], flavones [7], proteins [8–10], and other bioactive components from herb-based medicines [11,12], as well as polysaccharides [2,13] and uridine phosphorylase [14]. By contrast, ABS based on low-molecularweight (<1000) PEG has been rarely reported, because its higher affinity for water makes it difficult to form ABS. Martins et al. studied the ABS composed of PEG 400 and sulphate [15]. Al-Marzouqi et al. studied the hydrodynamics of PEG (300, 600, and 1000)phosphate ABS in CCC [16], but no actual sample was separated. Thus, ABS based on low-molecular-weight PEG remains a challenge for HSCCC separation. Furthermore, the establishment of the ABS used in HSCCC was not fully considered from the phase diagrams, phase forming time and phase ratio. The column volume, rotation speed, flow rate of the mobile phase, and separation temperature are important factors for HSCCC. The column volume cannot be changed in a fixed instrument, but other factors can be optimised. The effect of temperature on the phase diagrams is not significant [15], in fact, it is smaller with lowmolecular-weight PEG/salts ABS than with high-molecular-weight

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PEG/salts ABS [16]. Al-Marzouqi et al. found that higher stationary phase retention was achieved with the ABS of PEG300-phosphate than with the ABS of PEG600 (or PEG1000)-phosphate at a constant rotation speed and flow rate of mobile phase [16]. Hence, the ABS of low-molecular-weight PEG/salts is considered more advantageous for the separation of actual samples. Furthermore, the ABS of lowmolecular-weight PEG/salts possesses low viscosity [16], which is beneficial for the separation of high-viscosity substances. Pericarpium granati is widely used in traditional Chinese medicine, where it is frequently used to treat diseases such as diarrhoea and is also used as a haemostatic and as an insect repellent. Few researchers have studied the role of polysaccharide as one of the effective components of P. granati. As is well known, most polysaccharides from plants exhibit a variety of biological activities such as antioxidant [17], anti-tumour, anti-cancer [18,19], anti-inflammation [20,21], anti-glycation [22,23], and immunostimulatory activities [24]. The objective of the present work is to develop a new HSCCC system for separating polysaccharides from P. granati (PGP) using ABS based on low-molecular-weight PEG. 2. Experimental 2.1. Reagents and materials Crude PGP were prepared as described in a previous report [25]. Polyethylene glycols with an average molecular weight of 200, 400, and 600 (abbreviated as PEG200, PEG400, and PEG600, respectively) were obtained from SenBo Biotech Co. Ltd. (Xi’an, China). The reference substances of the monosaccharides and dextrans with different molecular weights were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and were acquired from Sinopharm Chemical Reagent Co., Ltd. Water was prepared from a Milli-Q water purification system (Millipore, MA, USA). 2.2. Selection of ABS Phase diagrams were used to establish the ABS. The binodal curves in the phase diagrams were determined by the cloud point titration method at 30 ± 0.5 ◦ C and atmospheric pressure, according to Ref. [26]. Different molecular weight PEGs and aqueous solutions of inorganic salts at 25% (w/w) were used for the phase diagram binodal determinations. Repetitive dropwise addition of the pure PEG liquid into a solution containing 25% inorganic salt under constant stirring was carried out until a cloudy solution was observed, followed by the dropwise addition of ultrapure water until a limpid and monophasic solution was obtained. The ternary system compositions were determined by the weight quantification of all components added within an uncertainty of ±0.1 mg, and the final mass fraction of each component was calculated [3]. The forming time of two phases was recorded, and then the upper and bottom phases were separated and the volumes were measured. The phase ratio was calculated as U/B, where U is the volume of the upper phase, and B is the volume of the bottom phase. The densities of the upper and lower phases were determined with a DMA 4500 density metre (Shanghai, China). The viscosity of each phase was measured using an SNB-2 digital viscosity metre (Shanghai, China). All physical property measurements were made in triplicate at 30 ± 0.5 ◦ C and atmospheric pressure. In order to obtain a satisfactory distribution coefficient (K) of PGP in the HSCCC separation and purification, the optimum solvent system was selected by measuring the K values in a series of PEG400/inorganic salt ABSs. About 1.0 mL of each phase of the PEG400/inorganic salt ABS and about 5 mg of the sample were

placed in a test tube, completely mixed, and then settled at the ambient temperature. After two clear layers were formed, the sugar contents in the upper and lower layers were measured by the phenol–sulphuric acid method [27], and the K value was calculated as K = MU /ML , where MU and ML represent the sugar contents in the upper and lower layers, respectively. 2.3. HSCCC separation procedure A model TBE-300A HSCCC system manufactured by Tauto Biotech Co. (Shanghai, China) was used for the separation of the PGP. A multi-layer coil planet centrifuge (CPC) was prepared by winding 2.5 mm i.d. PTFE tubing coaxially onto a column holder with a total capacity of 280 mL. The ˇ-value varied from 0.42 at the internal terminal to 0.63 at the external terminal. ˇ = r/R, where r is the distance from the coil to the holder shaft, and R is the revolution radius or the distance between the holder and central axes of the centrifuge. The rotation speed is adjustable from 500 to 1000 rpm, and the 750–900 rpm speed was used in the present study. In contrast to ordinary HSCCC systems, the system used in the present study was equipped with a thermostatic jacket. The jacket can keep the CPC at constant temperature, with the aim of eliminating the negative effect of temperature variations on the separation efficiency. Before each separation, the HSCCC column was first filled completely with the upper phase of ABS (i.e. the stationary phase), and then the apparatus was started at 800 rpm. At the same time, the lower phase of ABS (i.e. the mobile phase) passed into the column at a flow rate of 0.8 mL/min. After hydrodynamic equilibrium was reached, the crude extract sample (0.4 g) dissolved in a mixture of 20 mL each of the lower phase and upper phase was injected into the column through an injection loop. The effluent was collected with a DBS-100 mode fraction collector every 8 min and examined using phenol-sulphuric acid at 490 nm [27]. The fractions belonging to the same component were combined and dialysed with dialysis tubing (molecular weight cut-off of 2000 Da). After the salt and PEG were removed, each polysaccharide component solution was evaporated under vacuum, precipitated, and then dried to obtain the purified polysaccharide. 2.4. Determination of the average molecular weight and composition of the polysaccharide The determination of the average molecular weight was carried out by gel permeation chromatography (GPC) [7] equipped with a Waters 1515 Isocratic HPLC Pump System, Waters 2414 Refractive Index Detector, and Waters 717 Plus Autosampler. The data were processed by GPC processing software (Breeze). This experiment was performed with 0.1 M NaNO3 as the mobile phase at a flow rate of 0.6 mL/min. A sample at a concentration of 2 mg/mL was prepared and 110 ␮L of the solution was injected in each run. A set of T-series dextrans with different molecular weights (Mp = 0.61 × 104 , 0.96 × 104 , 2.11 × 104 , 4.71 × 104 , 10.7 × 104 , 19.4 × 104 , 34.4 × 104 , and 70.8 × 104 ) were used as reference substances to estimate the average molecular weight of the polysaccharides. The composition determination of the monosaccharides was conducted by gas chromatography–mass spectroscopy (QPGCMS2010, Shimadzu, Japan). The polysaccharide was first hydrolysed with 2.0 M TFA at 120 ◦ C for 2 h, and then the hydrolysate was conventionally converted into alditol acetates [28] and analysed by GC–MS. The detection procedure was carried out at the following heating profile: an initial temperature of 120 ◦ C, followed by heating at 5 ◦ C min−1 to 170 ◦ C, 1 ◦ C min−1 to 180 ◦ C, and then 5 ◦ C min−1 to 280 ◦ C. The flow rate of the helium carrier gas was 3.0 mL/min.

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The interface temperature was 280 ◦ C and the ion source temperature was 200 ◦ C. 2.5. Structure characterisation of the polysaccharide Fourier transform infrared spectroscopy (FTIR, EQUINX 55, Bruker Inc., Germany) was used to investigate the vibrations of the molecules and polar bonds between the different atoms and to characterise the structures of the polysaccharides. Infrared spectra (KBr) were obtained at the absorbance mode in the 400–4000 cm−1 region. After drying the polysaccharide in a vacuum drier for several days, it was dissolved in D2 O [29] for nuclear magnetic resonance (NMR) measurements. The deuterium-exchanged polysaccharide was put in a 5-mm NMR tube and the spectra were recorded with a Bruker Avance 500 spectrometer (Bruker Inc., Switzerland). The 1 H spectra were recorded at 50 ◦ C. 3. Results and discussion 3.1. Optimisation of the aqueous biphasic system A successful separation by HSCCC depends upon the selection of a suitable two-phase solvent system. To establish the ABS, some important factors, including PEG molecular weight and the type of inorganic salt, were investigated on the basis of the phase diagrams, phase ratio, and phase forming time. Fig. 1 depicts the effect of the molecular weight of PEG on the formation of ABS. The two-phase area (Fig. 1a) of the formation increases gradually with the increase in the degree of PEG

polymerisation in the PEG/MgSO4 ABS. This demonstrated that high-molecular-weight PEG easily forms an ABS, which agrees with the results of the previous report [15]. When the phase ratio (Fig. 1b) was 1, the phase-forming times (Fig. 1c) of the three ABSs were different from each other. PEG400/salt ABS needed the shortest time, which agrees with the known literature [3]. Therefore, the PEG400/salt was considered to be the preliminary optimum ABS. The effects of the cations and anions of the inorganic salt in the PEG400/salt ABS were investigated. As shown in Fig. 2, the phaseforming ability (Fig. 2a) of Na2 SO4 and MgSO4 were stronger than Al2 (SO4 )3 in the ABS formed by PEG400 and the salts with a common anion. It is recommended that the phase ratio (Fig. 2b) should be about 1 to avoid wastage of solvent and that the phase-forming time (Fig. 2c) should be shorter than 30 s to achieve good stationary phase retention in HSCCC [30]. Among the three ABSs, the ABS with Na2 SO4 needed the shortest time to induce the formation of the two phases. Therefore, Na2 SO4 was chosen as the optimum inorganic salt to form ABS. In order to further investigate the effect of the anion, Na2 SO4 was compared with NaNO3 and Na3 PO4 . According to the Hofmeister series, the chaotropic/kosmotropic characters of the various salts were different [3]. NaNO3 is a salting-in salt and, as a result, is not able to salt-out the polymer to form an ABS. As shown in Fig. 2d, a higher concentration of PEG400 was needed to form the PEG400/Na3 PO4 ABS because of the lower solubility of Na3 PO4 in water. Furthermore, the formation of the PEG400/Na3 PO4 ABS needed a longer time. Thus, the PEG400/Na2 SO4 system was possible as the optimum ABS. An optimum partition coefficient (0.5 < K < 2) for the targeted compounds is usually preferred for a successful HSCCC

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separation. The K values of the PGP in the three PEG400/inorganic salt (Na2 SO4 , MgSO4 , Al2 (SO4 )3 ) ABSs were further investigated and K values of 0.70, 0.66, and 0.78 were obtained, respectively. All the three systems have suitable partition coefficients; however, the shorter phase-forming time made the system with Na2 SO4 more favourable. Therefore, PEG400–Na2 SO4 –water was chosen as the best ABS for the HSCCC separation of PGP.

phase and 0.8 mL/min for mobile phase, in which the stationary phase retention was 34.62%. The elution profile (Fig. 3) was plotted by determining the contents of the polysaccharide components. Three fractions of polysaccharides were obtained: fractions 29–40, 53–58, and 67–97 corresponded to PGP-1, PGP-2,

3.2. HSCCC one-step separation of polysaccharides The density and viscosity of ABS composed of PEG400–Na2 SO4 –water were determined. The densities of the upper and lower phases were 1090 and 1289 kg m−3 , respectively, and the density difference and density ratio were −199 kg m−3 and 0.85, respectively. The viscosities were 83.2 and 42.0 mPa s of the upper and lower phases, respectively, and the viscosity difference and viscosity ratio were 41.2 mPa s and 1.98, respectively. From these results, it is concluded that the upper phase corresponds to the aqueous PEG-rich phase, while the lower phase is mainly composed of Na2 SO4 and water [31]. The upper phase was chosen as the stationary phase, while the lower phase was chosen as the mobile phase [16]. The separation temperature (25, 30, 35 ◦ C), rotation speed (700, 750, 800, 850, 900 rpm), and flow rate of the mobile phase (0.5, 0.8, 1.0, 1.5, 2.0 mL/min) affected the separation performance and were studied with the response index of the stationary phase retention. A higher stationary phase retention means that polysaccharides will show better separation. The optimum condition was operation temperature of 30 ◦ C, rotation speed of 800 rpm, and the flow rates of 10 mL/min for stationary

Fig. 3. HSCCC separation profile of PGP using the lower phase of the two-phase solvent (PEG400–Na2 SO4 –H2 O, 20%–16%–64%) as the mobile phase at a flow rate of 0.8 mL/min. The effluent was collected at 8 min/tube. The polysaccharide was examined by phenol–sulphuric acid spectrophotometry at 490 nm. The effluent was combined into three fractions: PGP-1 (tubes 29–40), PGP-2 (tubes 53–58), and PGP-3 (tubes 67–97).

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4. Conclusion An aqueous biphasic system based on low-molecular-weight PEG (PEG400–Na2 SO4 –H2 O) was first developed for the HSCCC separation of polysaccharides. This work demonstrated that combining phase diagrams, phase ratio, and time of phase forming with a partition coefficient is a very powerful tool for the selection of an HSCCC diphase solvent system. It is believed that the low-molecular-weight PEG aqueous biphasic system would offer potential advantages for the separation of bioactive substances. Acknowledgments The project was supported by National Natural Science Foundation of China (No. 21275098) and Doctor Base Foundation of Chinese Ministry of Education (No. 20110202110005). Fig. 4. FTIR spectra (KBr) of (a) PGP-1, (b) PGP-2, and (c) PGP-3.

References and PGP-3. The same polysaccharide fractions were combined and dialysed. After wiping off the salt and PEG, each polysaccharide solution was evaporated under vacuum and dried, and 10.7 mg of PGP-1, 4.7 mg of PGP-2, and 15.6 mg of PGP-3 were obtained. 3.3. Characterisation of the HSCCC fractions The GPC results indicated the homogeneity of PGP-1, PGP-2, and PGP-3. According to the retention time, the molecular weights were estimated to be 13,210, 2584, and 2459 Da, respectively, for PGP-1, PGP-2, and PGP-3. The GC–MS analysis showed that PGP1 was composed of xylose (12.4%), ribose (10.1%), and glucose (77.5%); mannose was the only component of PGP-2, and PGP-3 mainly comprised ribose (51.4%), mannose (26.7%), and glucose (21.9%). In the FTIR spectrum of PGP-1 (Fig. 4a), a strong band at 3428 cm−1 (hydroxyl group) was observed, together with bands around 2928 cm−1 (C H stretching vibration) and 1738 and 1626 cm−1 (the ester carbonyl and carboxyl groups), as well as strong absorbance at 1097 and 1427 cm−1 (attributed to the ˛-pyranose ring of the glucosyl residue) [32]. In addition, the characteristic absorption at 886 cm−1 of ˇ-sugars was observed. The strong signals at about ı 4.70 and ı 4.37 ppm in the 1 H NMR data of PGP-1 further confirmed the presence of the ˇ-configuration [13]. These results fully prove that PGP-1 mainly contains a ˇ-glycosidic bond, together with a partial ˛-configuration. Fig. 4b shows the FTIR spectrum of PGP-2; the strong band at 3439 cm−1 is owing to the hydroxyl group. The bands in the region 2922 cm−1 are owing to the C-H stretching vibration, and those at 1629 cm−1 are owing to the ester carbonyl and carboxyl groups. The bands at 1391 cm−1 are owing to the C-H variable angle vibration. The characteristic absorption at 884 cm−1 indicated that PGP-2 contains components with ˇ-configuration. Combined with the 1 H NMR data, the only strong signal of ı is obtained at 4.70 ppm; hence, it can be concluded that PGP-2 contains only ˇ-configuration. The IR spectrum of PGP-3 is shown in Fig. 4c; strong bands at 3440, around 2927, and 1430 cm−1 result from the hydroxyl group, C H stretching vibration, and C H in-plane flexural vibration, respectively. Bands at 1742 and 1629 cm−1 are caused by the ester carbonyl and carboxyl groups. The characteristic absorption at 890 cm−1 revealed that PGP-3 was a ˇ-sugar. The 1 H NMR data shows strong signals at ı 4.36 and ı 4.70 ppm, further confirming that PGP-3 was a polysaccharide with ˇ-configuration.

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Please cite this article in press as: X.-Y. Zhou, et al., Aqueous biphasic system based on low-molecular-weight polyethylene glycol for one-step separation of crude polysaccharides from Pericarpium granati using high-speed countercurrent chromatography, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.08.034