Structure elucidation of a pectin from Dendrobium nobile Lindl. and its immunological activity

ABSTRACT In this study, an RG-I pectin DNP-W6 was isolated from the stems of Dendrobium nobile Lindl. (Orchidaceae). It contained mannose, glucose, galactose, galacturonic acid and rhamnose in a molar ratio of 0.6: 1.0: 1.5: 1.1: 1.0. Its structural features were revealed by chemical, physical and spectral analysis. The results suggested that DNP-W6 possessed a backbone of a disaccharide of →4)-α-GalAp-(1→2)-α-Rhap-(1→, with approximate 50% substitution at O-4 of the rhamnopyranosyl residues. The side chains were neutral chains including galactosyl, mannosyl and glucosyl chains. The acetyl content was estimated to be 8.9%. The immunological results indicated that DNP-W6 and its derivatives exhibited different immunological activities in vitro, showing that the side chains and acetyl groups had an important effect on the expression of immunological activities.


Introduction
Pectic polysaccharides are a complex family of polysaccharides which occur naturally as structural elements in the primary cell walls and intracellular regions of higher plants [1,2]. The chemical structures can be divided into three main types: homogalacturonan (HG), rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) [2][3][4][5]. HG is a linear chain of (1!4)-a-D-galacturonic acid residues with some of the carboxyl groups methyl esterified [3]. RG-I contains a backbone of the repeating disaccharide [!4)-a-GalAp-(1!2)-a-Rhap-(1!] with substitution at O-4 of the Rhap residues [4]. RG-II is not structurally related to RG-I, since its backbone consists of alternating sequences of Rha and GalA rather than the repeating disaccharide [!4)-a-GalAp-(1!2)-a-Rhap-(1!] [5]. In addition, side chains of RG-II are attached to O-2/O-3 of GalAp and O-4 of the Rhap residues. The research on bioactivity and structure-activity relationship of pectin indicates that the pectic polysaccharides exhibit a variety of biological activities, such as anti-complementary, anti-tumor and immunological activities [6][7][8]. Moreover, the substituted groups play an important role in the expression of biological activities [9,10]. In the food industry, pectic polysaccharides are used as thickener, stabilizer, gelling agent, emulsifier, flavour fixation agent and texture modifier [1,11,12]. Pectic polysaccharides are widely studied due to their bioactivities and applications, such as pectin from Panax ginseng [13], Centella asiatica [4] and Diospyros kaki Thumb [14], etc. Dendrobium nobile Lindl. (Chinese name 'Jin-Chai-Shi-Hu') is a well-known edible and medicinal plant with wide distribution in the southwest and south of China. It is recorded in Chinese Pharmacopoeia [15]. Its stems are used as materials to make herbal tea or a functional beverage for thousands of years in China. In our laboratory, one pectin fraction (DNP-W6) was isolated and purified from D. nobile. In the present paper, we elucidate the structural features and pharmacological activities of DNP-W6, as well as its structure-activity relationship.

Analytical methods
The analytical methods used to determine the composition and structure were performed as previously described [7]. Briefly, homogeneity and molecular weight were determined by high performance gel permeation chromatography (HPGPC). Infrared (IR) spectra were recorded using a Perkin Elmer 599B FTIR spectrometer (Waltham, Massachusetts, USA). Gas chromatography (GC) analysis was done on a Shimadzu GC-9A instrument. Gas chromatography mass spectrometry (GC-MS) examination was performed on a Trace GC2010/Trace MS chromatograph. The 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded in D 2 O on a Bruker Avance DPX-400 instrument. The electrospray ionization mass spectrometry (ESI-MS) analysis of oligosaccharide fractions were performed on a VG Quattro MS/MS spectrometer as described in [16,17].

Isolation and purification of polysaccharides
The extractions of crude polysaccharide DNP-W referred to our previously reported method [7]. The dried D. nobile powder was extracted with distilled water. After precipitation, the supernatant was deproteined, dialyzed (3500 Da molecular weight cut-off) and lyophilized to give the crude polysaccharide DNP-W. DNP-W was subjected to a DEAE-cellulose column and eluted with 0.5 mol/L NaCl. Then, 0.5 mol/L NaCl eluation was further purified on a Sephacryl S-200 column to give DNP-W6. The eluate was monitored by the phenol-H 2 SO 4 method [18].

Chemical analysis
DNP-W6 (10 mg) was hydrolyzed with 2 mol/L TFA (4 mL) at 120 C for 4 h in a sealed tube. After removing the TFA in vacuum, the hydrolyzate was dissolved in water. One part of the hydrolyzate solution was used for thin layer chromatorgraphy (TLC) analysis. The other portion was reduced with NaBH 4 and acetylated with AcOH as previously described [7,19,20]. The resulting alditol acetates were analyzed by GC and GC-MS. In addition, another portion of DNP-W6 (20 mg) was reduced with CMC-NaBH 4 to give a carboxyl-reduced polysaccharide DNP-W6R [20]. Then DNP-W6R was hydrolyzed and analyzed as described for DNP-W6. The m-hydroxylbiphenyl method [21] was used to determine the uronic acid content of DNP-W6.

Methylation analysis
The samples of DNP-W6 and DNP-W6R were methylated according to the Ciucanu-Kerek method [22]. After complete methylation, the permethylated polysaccharides were hydrolyzed, reduced and acetylated. The resulting product was analyzed by GC-MS according to the method described by Sweet et al. [23].
Partial hydrolysis with acid DNP-W6 (500 mg) was treated with 0.15 mol/L TFA at 100 C for 40 min. The hydrolyzate solution was dialyzed against distilled water (3500 Da molecular weight cut-off). The solution inside the dialysis bag was concentrated and lyophilized to obtain a degraded polysaccharide (DNP-W6P, 210 mg). The exterior solution was collected and lyophilized (DP). DP was applied to a gel filtration chromatograph (Sephadex G-10 column) to give a series of oligomer fractions (DP1, DP2, DP3 and DP4) with increasing molar mass. The oligomer fraction DP4 was further hydrolyzed with 0.2 mol/L TFA at 100 C for 1 h to give a main degraded product DP5.

De-acetylation and determination of O-acetyl groups
The degree of acetylation (DA) in polysaccharides was assayed by applying a combination of 1 H NMR and the method described by Tomoda et al. [3,24,25]. According to the latter method, DNP-W6 (35 mg) was reacted with 0.3 mol/L NaOH (30 mL) at room temperature for 2 h. The solution was neutralized with HCl and dialyzed against distilled water. The resulting product was termed DNP-W6A. Then, DA of the degraded products was determined.

Immunobiological activity assay
The immunobiological activities of polysaccharide fractions were assayed according to the method described previously [7]. Spleen cells were isolated from male BALB/c mice and adjusted to a concentration of 5.0 £ 10 6 cells/mL with RPMI-1640 medium. After cell suspensions (100 mL) were seeded into a 96-well plate in the presence of mitogen ConA (5.0 mg/mL) or LPS (10.0 mg/mL), different dilutions of the polysaccharide samples (25 mg/mL, 50 mg/mL and 100 mg/mL) were added. Then, T and B lymphocyte proliferation was evaluated by the MTT method [26,27].

Data analysis
The data were represented as mean values with standard deviation ( § SD) of triple results. Student's t-test for unpaired observations between control and tested samples was used to identify statistical differences.

Results and discussion
Purification and structural characterization The dried stems of D. nobile were extracted three times with water and the extracts were precipitated with 4 times the volume of 95% ethanol to precipitate the crude polysaccharide DNP-W. After fractionation on DEAE-cellulose chromatography, a fraction was obtained from 0.5 mol/L NaCl eluate. Then it was further purified by Sephacryl S-200 gel filtration chromatography and gave a homogeneous polysaccharide DNP-W6 confirmed by HPGPC (Figure 1). The average molecular weight of DNP-W6 was estimated to be 6.6 £ 10 5 Da. Its specific rotation [a] D 20 is ¡7.1 (c 0.80, H 2 O). DNP-W6 was free of protein according to the Lowry method and gave no absorption at 280 nm [28]. The IR spectrum of DNP-W6 is shown in Figure 2. The bands at 3421, 2914 and 1646 cm ¡1 were due to the hydroxyl stretching vibration, C-H stretching vibration and associated water, respectively [29]. The peaks at 810 cm ¡1 were assigned to the D-galactopyranose unit. The clear absorption at 1744 cm ¡1 indicated the presence of uronic acid groups in DNP-W6 [30]. The uronic acid content was calculated to be 18.6% by the m-hydroxybiphenyl method [21].
The sugar components of DNP-W6 were determined by TLC and GC analysis. TLC analysis showed that DNP-W6 contained mannose, glucose, galactose, rhamnose, as well as some amount of galacturonic acid. After DNP-W6 was reduced three times with CMC-NaBH 4 , a carboxyl-reduced polysaccharide DNP-W6R was obtained. A compared analysis of DNP-W6 and DNP-W6R by GC indicated that DNP-W6 was composed of mannose, glucose, galactose, galacturonic acid and rhamnose in a molar ratio of 0.6: 1.0: 1.5: 1.1: 1.0 ( Table 1). The ratio of GalA was obtained by computation of the difference in the content of Gal in DNP-W6 and DNP-W6R.
The signal at d 2.01 ppm in the 1 H NMR spectrum (Figure 3) indicated the presence of O-acetyl groups. The content of O-acetyl groups was calculated to be 8.9% according to 1 H NMR and the Tomoda method [24].
Signals in the 1 H NMR and 13 C NMR spectra of DNP-W6 were assigned insofar as possible based on the above results and published data [31][32][33]. The 1 H NMR spectrum of DNP-W6 is shown in Figure 3. The signals at      (Table 1) showed that partial Gal residues, Man residues and Glc residues were removed. But compared with these changes, Rha and GalA residues were largely retained, indicating pectic component RG was the core structure of DNP-W6. The methylation analysis of DNP-W6P (Table 2)

ESI-MS analysis
DNP-W6 was treated with TFA to give a series of hydrolytic fractions (DP1, DP2, DP3 and DP4) by Sephadex G-10 column according to increasing molar mass. The composition of DP1 was a mixture of monosaccharides. DP2 and DP3 were very complex and contained much more components. Thus they were not investigated further. DP4 was a main eluate fraction of dialysate. According to the TLC and GLC analyses of DP4, it mainly contained Rha, GalA, Gal, and Glc residues (Table 1). ESI-MS experiments of DP4 were carried out. The negative ESI-MS spectrum of DP4 is shown in Figure 5

Immunological activity and structure
The in vitro immunological activities of DNP-W6 and its derivatives were evaluated and compared ( Table 3). The pharmacological assay indicated that DNP-W6 could stimulate the proliferation of ConA-or LPS-induced lymphocytes even at the 25 mg/mL level. The activity followed a dose-dependent response curve. The hydrolyzate DNP-W6P enhanced the proliferation of ConA-induced lymphocytes only at 100 mg/mL dose, suggesting that the side chains of DNP-W6 had an effect on the expression of immunological activity. In addition, DNP-W6A, the products of deacetylated DNP-W6, showed the immunoenhancing activities of ConA-or LPS-induced lymphocytes also only at 100 mg/mL dose, indicating that the acetyl groups played an important role in lymphocytes. The results above suggested carboxyl and acetyl groups played an important role in the properties of DNP-W6.
The molecular weight, monosaccharide composition, functional group, branching degree and glycosidic  linkage are some of the structural features recognized to contribute to the polysaccharides immunostimulatory activity [34]. The content of uronic acids has been considered to play important roles in biological activity [35][36][37], and many acid polysaccharides have immunomodulatory activity [36][37][38]. The existence of functional groups of monosaccharides is a crucial structural feature for immunostimulatory activity [36,37]. However, the structural features of specific polysaccharides may impact the resulting activity in different ways. The effect of the functional group of pectin from D. nobile on the immunological activity has been poorly studied. To the best of our knowledge, this paper investigated, for the first time, the relationship between acetyl groups in polysaccharide from D. nobile and the immunostimulatory activity. The obtained results are useful in enhancing the knowledge of the structure-activity relationship of pectin, as well as for expanding the use of D. nobile.

Conclusions
The present study revealed that a novel polysaccharide (DNP-W6) with an average molecular weight of 6.6 £ 10 5 Da was isolated from Dendrobium nobile Lindl. TLC and GC analysis indicated that DNP-W6 is composed of mannose, glucose, galactose, galacturonic acid and rhamnose in a molar ratio of 0.6: 1.0: 1.5: 1.1: 1.0. Spectral analysis concluded that DNP-W6 is complex pectin. Immunological activity analysis suggested that the side chains, carboxyl and acetyl groups had effect on the expression of immunological activity. These findings are helpful for the potential application of DNP-W6, as well as for expanding the use of D. nobile.