Isolation, Purification and Structural Characterization of a Polysaccharide from Rheum Palmatum L

Rhubarb is a widely used Traditional Chinese Medicine in treating various diseases for thousands of years, and polysaccharide is one of the bioactive constituents in this herb. However, study of the polysaccharide is very limited, especially structural characterization. In the present study, a polysaccharide from Rheum palmatum L. (PR) was isolated and puried. Based on UV, IR, GC, GC-MS, 1H NMR and 13C NMR analyses, the structure of PR was characterized. Its molecular weight was measured to be 1.21 × 10 4 Da. The results of PR structural analysis showed that four 1→4 galactose were linked with one 1→ 3, 6 galactose and one 1→6 glucose, and each repeating unit had a branched chain consisting of a α-1→ xylose, which was linked with the C-6 of galactose backbone chain.


Introduction
Rhubarb (Da-Huang in Chinese medicine) is one of the most important herbal medicines that has been widely used for thousands of years in many Asian countries [1,2]. The dried roots and rhizomes of Rheum palmatum, R. tanguticum and R. offcinale are officially recorded by the Chinese Pharmacopoeia and European community monograph [1]. According to the principles of traditional Chinese medicine, rhubarb is frequently used in clinical treatment for its wide range of therapeutic activities, such as analgesic, choleretic, purgative, antibacterial, antiplatelet, anti-cancer, anti-inflammatory, antioxidant, liver-protective activities [3][4][5]. These pharmacological effects are attributed to components isolated from rhubarb, including anthraquinone derivatives, anthocyanin, polyphenols, polysaccharide and stilbenes [2,6,7]. To the best of our knowledge, secondary metabolites in the rhubarb have been widely studied by numerous researchers [6,8]. However, there are few reports on the structure and conformation of polysaccharides.
Polysaccharides are macromolecular carbohydrates formed from long chains of monosaccharides and their main functions include energy storage, structural support, antigens, and so on [9] Recently, extensive attention has been paid to polysaccharides due to their crucial physiological and biological functions, such as anti-coagulant, cardiac protection, immunomodulatory and antitumor [10,11]. According to previous reports, the biological activities of polysaccharides were closely related to their chain conformation and structure including chemical compositions, molecular weight, backbone and degree of branching [12]. In this regard, the elucidation of structure and conformation is necessary to better understand biological functions of polysaccharides. Herein. A polysaccharide from R. Palmatum (RP) was isolated and purified, and its structural characteristics were analyzed comprehensively by a variety of analytical techniques such as UV, IR, GC, GC-MS and NMR.

Extraction of Crude Polysaccharide
Crude powder of R. palmatum was weighed for 300g accurately, and added 1L petroleum ether-absolute ethyl alcohol (1:1 for volume) to reflux degreasing for 2 h. Volatile petroleum ether was removed from the residue, and added 1L water to reflux extracted 3 times for each time with 2 h. The extracting solutions were combined and concentrated to 100 mL. The concentration was added ethanol to ethanol containing of 80%, and put at 4°C for 12 h, and centrifuged to obtain the precipitate. The precipitate was washed with diethyl ether and acetone several times respectively, and dried in low temperature to obtain the crude RP. The obtained crude polysaccharide was dissolved by water and added 3% papain, and put at 60°C for 3 h. The solution was removed proteins by Sevag method.

Purification of Polysaccharide
The crude polysaccharide was re-dissolved with 5 mL water, and purified by AB-8 (4 cm × 80 cm) macroporous resin column, which eluted by water with flow rate of 2ml/min, collected and concentrated eluant. The eluant was further purified by SephadexG-100 gel filtration chromatography (4 cm × 80 cm), eluted by water with flow rate of 2mL/min. The eluant was collected by tubes respectively (5 mL per tube), and measured the absorbance by phenol-sulfuric acid method at 490 nm. Combining the eluant, concentrating and freeze-dried were performed to obtain RP.

Measurement of Molecular Weights
The Polysaccharide was dissolved into the concentration of 2.0 mg/mL by water, and then filtrated by millipore filter and measured on a GPC using a Waters 1525 instrument. The purity and relative molecular weight of RP were determined on HPLC by using Waters series (Waters Ultrahydrogel TM 1000 column; ELSD detector; the temperature of column was 30°C; the mobile phase was 0.05% mixed solution of phosphoric acid-water; isocratic elution; injection volume was 20 µL; flow rate was 0.4 mL/min). Dextran T-12.6, T-60, T-110, T-300, T-2000 and glucose were as standard [13].

NaIO 4 Oxidation
The polysaccharide (25.0 mg) was dissolved in 25mL 15 mM NaIO 4 solution and reacted in the dark condition. The solution was sampled every 6 h and followed by spectroscopy at 223 nm. Ethylene glycol was added to terminate the reaction when the consumption of IO 4 was stable. Consumption of IO 4 was calculated according to the standard curve that was made with NaIO 4 solution at 223 nm. The content of formic acid was determined by titration using 0.01 mol/L NaOH solution.

Monosaccharide Composition Analysis
50.0 mg polysaccharide was dissolved in 4.0 mL 2 mol/L trifluoroacetic acid (TFA) in a 10 mL test tube. The test tube was heated in water-bath at 100°C for 3 h to hydrolyze the polysaccharide to monosaccharides. The hydrolyzed solution was cooled to room temperature, and centrifuged for 8 min in 6000 r/min. The supernatant was collected and evaporated to dry under reduced pressure. The dried RP was dissolved in 600 µL hydroxylamine hydrochloride solution and then mixed by supersonic method for 1 min. The mixed solution was used to carry on the water bath for at 90°C 30 min, and cooled to room temperature. Then, 600 µL acetic anhydride was added to the cooled solution to carry on water bath at 90°C for 30 min again, cooled to room temperature. The solution was mixed with 500 µL chloroform, and next washed three times by 500 µL deionized water. The organic portion was collected and removed the excess water with anhydrous Na 2 SO 4 . Finally, the resultant solution was evaporated to dryness and re-dissolved in 500 µL chloroform for GC analysis. D-Glucose, D-galactose and D-xylose were also derivatized as standards. The analysis was performed using an Agilent 6890N chromatograph (USA) equipped with a capillary column of DB-5 (30 m × 0.25 mm × 0.25µm); The GC was run using the following conditions: injection temperature, 270°C; detector, flame-ionization detector; the temperature of detector, 270°C; the carrier gas, N2 at a flow rate 30 mL/min; sample injection was as the splitless method; the temperature program was that initial temperature was 150°C for 3 min, and as the rate of 3°C/min to 170°C, and holding for 5 min, and then as the rate of 5°C/min to 210°C, and holding for 5 min, and then as the rate of 10°C /min to 270°C, and holding for 5 min.

Methylation Analysis
Methylation analysis was carried out based on the previous method [14]. 10 mg RP which was sufficiently dried by P 2 O 5 was depolymerized the methylated products and hydrolyzed by trifluoroacetic acid, and then acetylated to generate alditol acetate correspondingly. For speculating the branch structure, RP was further analyzed by partial acid hydrolysis method. RP was hydrolyzed by 0.05, 0.2 and 0.5 mol/L trifluoroacetic acid respectively, and evaporated to dry under reduced pressure. RP was transferred to a dialysis bag after removed the trifluoroacetic acid, and dialyzed by ultrapure water for 48 h to obtain the high molecular weight retentate RP-a and low molecular weight permeate RP-b respectively. The solution was dried and treated with methylation process, and then analyzed by GC-MS. GC/MS analyses were conducted using a Shimadzu GC/MS QP2000 system under the same GC conditions. An electron impact quadropolar system was used in the detection.

Infrared Spectra
2 mg polysaccharide was processed by pressed-disk technique for Infrared spectra analysis A Fourier transform infrared spectropho-tometer (PerkinElmer Spectrum 400 FT-IR, Massachusetts, USA) in the range of 4000-500 cm -1 were applied [15].

NMR Spectra
12 mg RP was dissolved in 400 µL D 2 O for the analysis of NMR. The spectrogram of 1 H NMR and 13 C NMR were obtained with the following condition. 1 H NMR analysis, the frequency, 600 MHz; the temperature, 25°C. 13C NMR analysis, the frequency, 125 MHz; the temperature, 30°C. TMS was used as the internal standard. Crude polysaccharide (31.25 g) was extracted from crude powder of R. palmatum by water-extraction and alcohol-sedimentation method, and then crude polysaccharide was further purified by AB-8 macroporous resin and Sephadex-G 100 column chromatography. As a result, 1.05 g pure polysaccharide (RP) was obtained. The absorbance was measured at 490 nm by phenol-sulfuric acid method. The elution curves were shown in Figure 1. As showed in Figure 2, GPC chromatogram showed that the chromatographic peak of RP was single symmetrical peak of a homogeneous component. Based on the standard curve which was calculated by dextran (Y=12.171-0.4476X, r=0.9996) and the retention time of samples, the relative molecular weight of RP was determined to be 1.21 × 10 4 Da. The UV scanning in 260nm and 280nm wavelength shown that RP had no absorption, and the ninhydrin reaction was negative, which indicated that RP did not contain nucleic acid and protein.

NaIO 4 Oxidation Reaction
The glycosidic linkage locations of RP were preliminarily determined by consumption of NaIO4 and production of formic acid. The results showed that 1 mol of sugar residues consumed 0.64 mol of NaIO4 and produced 0.08 mol of formic acid. Meanwhile, the consumption of NaIO 4 was two times higher than the production of formic acid, which indicated that RP contained linkages which could produce formic acid such as (1→6, 1→) linkage, but also contained some amount of linkage types which only could consume NaIO4 such as (1→4) linkage or some amount of linkage types which could not be oxidized by NaIO 4 such as (1→3, 6) linkage.

Monosaccharide Composition Analysis
In order to obtain more information about the monosaccharide residues of RP, the product of NaIO 4 oxidation was degraded by Smith degradation method and then analyzed by GC. The chromatograms of standard monosaccharides and degradation products were shown in Figure 3. The results indicated that RP was mainly consist of galactose as well as a small amount of glucose and xylose, whose molar ratio was 5.1: 1: 1.

Methylation Reaction
To further determine the linkage types between monosaccharide residues, RP was permethylated, hydrolyzed and converted into partially methylated alditol acetates for GC-MS analysis. The results were shown in Table 1, the backbone structure of the sugar chain was mainly consist of 1→4 linked galactose (Gal), 1→ terminal xylosel (Xyl) and 1→3, 6 linked galactose (Gal), small amounts of 1→6 linked glucose (Gal), 1→3 linked glucose (Glc) and galactose (Gal). In Mass spectrum, m/z 43 was the base peak of methylated acid sugar esters (PMAA), and was formed due to the loss of acetyl ion (CH3CO+). The m/z 145 and 259 fragments were the characteristic peaks of hexose mass spectrum, while the erythritol and glycerol were generated.  The further research on branch structure of RP was shown in Table 2. After partial acid hydrolysis, the macromolecular fragment RP-a was the RP backbone structural components which consisted of galactose and glucose. Thus, the backbone structure of RP was composed of galactose and glucose alternately. The analysis by GC-MS for RP-b was shown to be 1→ terminal xylose. Because the RP-b was the lower molecular weight part with partial acid hydrolysis, so it was hydrolyzed as a branched chain of RP.

Infrared Spectroscopy Analysis
To characterize polysaccharide fractions, the characteristic absorption of RP was performed in the range of 4000-400 cm -1 by IR. The result was shown in Figure 4. It is indicated that distinct polysaccharide characteristic bands appeared at 3435, 2951, 1648, 1617, 1413 and 1101 cm -1 . The wide and strong absorption band at 3435 cm -1 was the O-H stretching vibration of sugar; resulting from intermolecular and intramolecular hydrogen bonding [16]. A weak peak at 2951 cm -1 was caused by C-H asymmetric stretching vibration; The peak at 1617 cm-1 was caused by the H-O-H bending of water adsorbed on the polysaccharide sample; The peak near 1413 cm-1 was caused by the vicinity of deformation vibration of CH 2 ; the peak near 1101 cm -1 1was attributed to the stretching vibration of C-O-C and the deformation vibration of O-H in C-O-H bond [17]. 955 cm -1 was caused by the symmetric stretching vibration of the furan ring; 779 cm -1 was caused by the stretching vibration of the pyran ring. Hence, monosaccharide residues in RP was appeared in form of furan ring and pyran ring. The result is consistent with monosaccharide composition analysis.

NMR Analysis
The structural feature of RP was also elucidated by NMR spectral analysis. The spectrums were shown in Figure 5. The chemical shifts at δ 4.30-5.90 ppm in the 1 HNMR spectrum could be assigned to the typical signals of anomeric protons. In the spectrum, three main signals at δ 5.47, 5.14 and 4.47 in the anomeric proton region indicated that RP was mainly composed of three types of monosaccharides. Among them, one signal at δ 4.47 was assigned to the configuration of β-glycosidic bond and others were assigned to the configuration of α-glycosidic bond. The strong signal at around 4.8 ppm was attributed to the chemical shifts of D 2 O. Correspondingly the 13 C NMR spectrum showed three carbon signals of δ 104.966, 100.244 and 93.454 in anomeric region, which were assigned to β-xylose, α-galactose and α-glucose, respectively. This is consistent with those obtained in GC and GC-MS analysis. In the non-anomeric carbon region, a large number of signals accumulated in the range of δ 61~77. The absorption peak in δ 82.6 indicated that the substitution occurred on C3 of glucose; the appears of signal δ79.19 indicated that substitution occurred on C4 of galactose residues; the strong absorption peak of signal δ 82.6 indicated that the substitution occurred on C3 of glucose residue; the presence of signal δ 63.6 indicated that sugar chain contained fructofuranosyl units; the strong absorption peak at δ 62.6 indicated that C6 was not substituted in most glucose.
The combined results from the analysis of NaIO4 oxidation, Smith degradation, IR, GC-MS and NMR indicated that the backbone chain of RP was composed of galactose linked by 1→4 and 1→3,6 glycosidic bonds as well as glucose linked by 1→6 glycosidic bonds, and the branch chains were attached to backbone chain by xylose linked by 1→ terminal glycosidic bonds. Namely, four 1→4 galactose were linked with one 1→ 3, 6 galactose and one 1→6 glucose, and each repeating unit had a branched chain consisting of a α-1→ xylose, which was linked with the C-6 of galactose backbone chain.

Conclusion
In conclusion, a polysaccharide from R. Palmatum (RP) was obtained in this study. The relative molecular weight of RP was 1.21 × 10 4 . Through structural characterization analysis, it is demonstrated that the backbone chain of RP is composed of glucose linked by α-(1→4) galactose, α-(1→3,6) galactose and α-(1→6) glucose, the branch chains are attached to backbone chain by β-1→terminal xylose. RP was built up of β-xylose, α-galactose and α-glucose residues in the molar ratio of 1:5.1:1. This study provided useful information for further study of the polysaccharide from R. palmatum.