Structural Characterization of Mannoglucan from Dendrobium nobile Lindl and the Neuritogenesis-Induced Effect of Its Acetylated Derivative on PC-12 Cells

A water-soluble polysaccharide (JCS1) was isolated from the stems of Dendrobium nobile Lindl. JCS1 was structurally characterized using a combination of chemical and spectral analysis, including methylation analysis, partial acid hydrolysis, Fourier-transform infrared (FTIR) spectroscopy, gas chromatography (GC), GC-mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. The molecular weight was estimated to be 2.3 × 104 Da using high-performance gel permeation chromatography (HPGPC). The sugar composition analysis indicated it was composed of glucose, mannose, xylose, and arabinose in a 40.2:2.3:1.7:1.0 molar ratio. The structure analysis showed that JCS1 was a mannoglucan with a backbone consisting of (1→4)-linked β-Manp and (1→4)-linked α-Glcp with branches at C-6 of (1→4)-linked α-Glcp residues. The branches were composed of T-α-Glcp, 1,4-α-Xylp, and T-α-Araf. In vitro bioactivity tests revealed that the acetylated derivative of JCS1, YJCS1, induced neuritogenesis of PC-12 cells. These results demonstrate that YJCS1 might be a promising bioactive polysaccharide for development as a drug candidate for the possible prevention and treatment of neurodegeneration diseases.


Introduction
Dendrobium nobile Lindl belongs to the orchid species and is widely distributed in Asia, including Thailand, Laos, Vietnam, and China [1,2]. It has been used as a medicinal and edible plant for thousands of years in China [2]. Its dried stems, the main medicinal part, have been studied for its "reinforcement" of body fluids, blood nourishment, promotion of saliva secretion, fever reduction, treatment of chronic gastritis, depression of cholesterol levels, and immunostimulation [2,3]. Recently, polysaccharides isolated from D. nobile have been demonstrated to possess immunostimulatory, antitumor, antioxidant, and hypoglycemic activities [3][4][5][6]. Attempts have been made to apply appropriate methods to modify the native polysaccharides to optimize their biological activities [7][8][9][10][11][12][13]. However, it is not clear whether acetylated modification of the native polysaccharide could induce neuritogenesis. In this paper, we

Preparation of JCS1
The herbals of D. nobile (2 kg) were cut into 2 cm piece of stem and defatted by immersion in 95% ethanol (40 L) for twice, each for five days, at 25 • C. The ethanol extract were stirred once every 1 h. Five days later, the herbal residues were immersed into 40 L of ethanol again to further defatted with stirring as aforementioned. After ethanol was removed, the ethanol-insoluble residues were air dried at 25 • C. Then the residues were extracted with boiling water (40 L) seven times, 4 h each time. After filtration, the extracts were concentrated to the volume of 4 L by heating at 100 • C followed by dialysis with filter bag (cutoff is 5000 Da) against running tap water for two days. After the dialyzed extract solution was centrifuged (8000 rpm) for 5 min, the supernatant was concentrated and precipitated with four volumes of 95% ethanol with vigorous stirring. After standing the mixture overnight, the mixture was centrifuged (8000 rpm) for 5 min, the precipitate was washed successively with absolute ethanol and acetone for three times, and then dried at 40 • C in an oven to obtain the crude polysaccharide, designated as JCS. JCS (10 g) was dissolved in 200 mL distilled water, and centrifuged (8000 rpm) for 5 min. The supernatant was fractionated using a Q Sepharose Fast Flow column (0.98 L) and eluted with 1.2 L of 0.1 M sodium chloride (NaCl) solution to obtain the target polysaccharide, JCS1.

Preparation of Acetylated Derivative
The JCS1 was acetylated using a previously described method [14]. Briefly, JCS1 (100 mg, dried under vacuum conditions overnight) was dissolved in 8 mL dimethyl sulfoxide (DMSO), stirred with a magnetic stirrer, and reacted for 24 h at room temperature to obtain a homogeneous solution. Then, 0.6 mL pyridine and 0.5 mL acetic anhydride was added sequentially to the reaction solution, the reaction was run for 2 h at 4 • C, and then it was terminated by adding distilled water, followed by dialyzation and lyophilization to obtain the acetylated derivative, YJCS1.
The acetyl group and degree of substitution (DS) of YJCS1 were determined using a previously reported method [14]. The DS values were calculated as follows: where M = acetyl group (%, expressed as a percentage of the detected JCS1).

Homogeneity and Molecular Weight
The homogeneity and molecular weight were determined using a high-performance gel permeation chromatography (HPGPC) method using an Agilent 1260 Series HPLC system (Santa Clara, CA, USA) with a tandem KS-804 and KS-802 (ID 8 mm and length 300 mm, Shodex Co., Tokyo, Japan). The column temperature was at 40.0 ± 0.1 • C. The mobile phase was 0.2 M NaCl run at a flow rate of 0.8 mL/min. All samples were prepared as 0.2% (w/v) solutions in the eluent, centrifuged, and then the supernatant was analyzed using a 20 µL injection volume in each run [15].

Monosaccharide Composition Analysis
The monosaccharide composition was analyzed according to a previous method [15]. Briefly, samples (2 mg) were dissolved in 2 mL water (H 2 O) and hydrolyzed with the same volume of 4 M trifluoroacetic acid (TFA) in a sealed test tube at 110 • C for 4 h. The solution was repeatedly evaporated with methanol to completely remove the TFA under reduced pressure. The hydrolysate was dissolved in 2 mL H 2 O and reduced with 50 mg sodium borohydride (NaBH 4 ) for 3 h at room temperature while shaking the sealed test tube occasionally. The reduction reaction was neutralized with 25% acetic acid and evaporated with methanol to obtain a dry powder, which was further dried in an oven at 100 • C for 30 min. Then, the dried residue was acetylated with 3 mL acetic anhydride (Ac 2 O) for 1.5 h at 100 • C. After evaporating to dryness with toluene, the residue was extracted with chloroform (CHCl 3 ), washed three times with distilled H 2 O (1:1, v/v), and the resulting alditol acetates were analyzed using gas chromatography (GC). The GC analysis was performed using a Shimadzu GC-14B instrument (Kyoto, Japan) with a 3% OV-225-packed glass column (3.2 mm × 200 cm). The detector was flame ionization detector (FID, Kyoto, Japan). The temperatures were 250 and 240 • C for injection and detection, respectively. The flow rate of the nitrogen carrier gas was 25 mL/min, and the column temperature was maintained at 210 • C.

Methylation Analysis
Using a previously reported method [16], the vacuum-dried polysaccharide (10 mg) was methylated three to four times until the polysaccharide hydroxyl absorption in the infrared (IR) spectrum (Nujol) disappeared. Then, the methylated polysaccharide was hydrolyzed with 2 M TFA at 110 • C for 4 h, reduced with NaBH 4 , and finally acetylated to obtain the partially methylated alditol acetates, which were analyzed using GC-mass spectrometry (MS, HP-5 capillary column, 0.25 mm × 30 m, Santa Clara, CA, USA). The carrier gas was nitrogen, and the column temperature was gradually increased at 2 • C/min from 140 to 250 • C.

Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) Analysis
To determine the polysaccharide FT-IR spectra, the samples were first prepared in two forms. For example, the native and per-methylated polysaccharides were processed as potassium bromide (KBr) pellets and Nujol films, respectively. Furthermore, the FT-IR measurement (PerkinElmer 591B, Waltham, MA, USA) was performed in the frequency range of 4000-500 cm −1 .
The 1 H NMR, ( 1 H)-1 H correlation spectroscopy (COSY) and 13 C NMR (heteronuclear single-quantum correlation [HSQC] and heteronuclear multiple-bond correlation [HMBC]) spectra were recorded at 25 • C using a Bruker AVANCE III NMR spectrometer (Karlsruhe, Germany) with acetone as the internal standard, conducted at 500 and 125 MHz, respectively. For the deuterium-exchange, the polysaccharides (30 mg) were dissolved in 0.5 mL D 2 O (99.9% D). Furthermore, 31.50 and 4.85 ppm for the 13 C NMR (acetone) and 1 H NMR (HDO), respectively, were used as the references to calibrate the chemical shifts.

Partial Acid Hydrolysis
The JCS1 (100 mg) was dissolved in 0.05 M TFA (10 mL), hydrolyzed at 100 • C for 1 h, the resulting solution was evaporated to dryness, and then dialyzed for 48 h. The collected retentate (60 mg) was further hydrolyzed with 0.1 M TFA (6 mL) at 100 • C for 1 h. Homogeneity, molecular weight, and monosaccharide composition, as well as methylation and NMR analyses of the hydrolysates (JCS1 0.1N), were performed.

Bioactivity Test of Polysaccharide in PC-12 Cells
The PC-12 cells (ATCC) were seeded into 12-well plates containing 50 µg/mL polylysine for 24 h, and then they were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 1.0% horse serum and 0.5% fetal bovine serum (both serum are inactivated at 56 • C for 30 min before using) for 24 h. Subsequently, varying concentrations of polysaccharide JCS1 and YJCS1 were added to the cell culture medium and incubated for 72 h while 25 ng/mL nerve growth factor (NGF, Invitrogen, Boston, MA, USA) was used as a positive control. The cell morphology was observed using an inverted fluorescent microscope (Olympus IX73, Tokyo, Japan), and the images were acquired at a magnification of 400×. The experiments were performed in triplicate using duplicate wells.

Isolation, Purification, and Composition Analysis
The crude polysaccharide JCS was obtained in 1.3% yield by boiling-water extraction from the dried stems of D. nobile, and further fractionated by anion-exchange chromatography on a Q Sepharose Fast Flow column to give 0.1 M NaCl eluent JCS1 (yield 18.9%). JCS1 was demonstrated to be a homogeneous polysaccharide by the presence of a symmetrical peak on HPGPC. The average molecular weight of JCS1 was estimated to be 2.3 × 10 4 Da. The results showed that JCS1 contained 6.6% protein using the Lowry method [17], and was shown to be free of uronic acid using the m-hydroxyl diphenyl method [18]. The sugar composition of JCS1 was determined using GC analysis. The results showed that JCS1 contained glucose, mannose, xylose, and arabinose in a molar ratio of 40.2:2.0:1.3:1.0.

IR and Specific Rotation Analysis
The previously reported method used for the analysis [14] showed that JCS1 was acetylated and named YJCS1. The IR spectra of JCS1 and its acetylated derivative YJCS1 are shown in Figure 1. Typical polysaccharide signals were observed in their IR spectra. Compared with JCS1, the hydroxyl stretching band at 3431.7 cm −1 of YJCS1 was obviously smaller. The new absorption signals that appeared at 1247.6 and 1736.9 cm −1 , assigned as the C=O stretching vibration, indicating that the acetylated modification of JCS1 was successful [19]. The specific optical rotation of JCS1 was estimated to be 115.
Typical polysaccharide signals were observed in their IR spectra. Compared with JCS1, the hydroxyl stretching band at 3431.7 cm −1 of YJCS1 was obviously smaller. The new absorption signals that appeared at 1247.6 and 1736.9 cm −1 , assigned as the C=O stretching vibration, indicating that the acetylated modification of JCS1 was successful [19]. The specific optical rotation of JCS1 was estimated to be 115.3° (c 1.0, H2O).

Partial Acid Hydrolysis and Structure Characterization of Degraded Polysaccharide
To characterize the JCS1, it was partially hydrolyzed with 0.05 M TFA and dialyzed to obtain the retentate, which was further hydrolyzed with 0.1 M TFA and dialyzed against water to provide the final retentate, named JCS10.1N. HPGPC analysis showed that JCS10.1N was homogeneous with a molecular weight of 15.5 kDa. The results of the monosaccharide composition analysis showed that JCS10.1N was composed of glucose, mannose, and xylose in a molar ratio of 40.1:2.2:1.0. It was methylated and analyzed using GC-MS, and the results are shown in Table 1. The results showed that the residues linkage types were 1,4-linked Glcp (17.8%), 1,4,6-linked Glcp (3.0%), 1,4-linked Manp (2.3%), and 1,4-linked Xylp (0.6%), and T-linked α-Glcp (2.6%). Compared with the methylation analysis of the native JCS1 (Table 1), the T-linked α-Araf vanished, which suggests that the Ara residues were sensitive to this mild acid. This indicates that Ara was probably located on the outer branches and that JCS1 has a (1→4)-linked Manp and (1→4)-linked α-Glcp backbone with branches at the C-6 of (1→4)-linked α-Glcp residues.

NMR Results
The 13 C NMR spectra of JCS10.1N and JCS1 are shown in Figure 2A,B, respectively. In the 13 C NMR spectrum of JCS1, the anomeric signals of Glc, Man Xyl, and Ara were assigned and combined with the 13 Figure 3B). Similarly, the signals at 4.55, 5.03, and 5.14 ppm were ascribed to the H-1 of 1,4-linked β-Manp, terminal α-Glcp, and 1,4-linked α-Xylp, respectively ( Figure 3A). The resonances at 5.11 and 5.24 ppm were attributed to the anomeric hydrogen of terminal α-Araf at different chemical locations. The other resonances were also assigned, and the chemical shifts corresponding to H2 to H5 or H6 of all the residues are listed in Table 2. were also assigned, and the chemical shifts corresponding to H2 to H5 or H6 of all the residues are listed in Table 2.    In the HMBC spectrum ( Figure  Based on the monosaccharide analysis, methylation, partial hydrolysis, and NMR analysis, a possible repeating unit for JCS1 was proposed as follows: different chemical locations and H-4 of 1,4-linked α-Xylp. According to the above results, the backbone of JCS1 might consist of repeated 1,4-linked β-Manp and 1,4-linked α-Glcp units with branches at the C-6 of 1,4-linked α-Glcp substituted by 1,4-linked α-Xylp and T-α-Araf linked at C-4 of 1,4-linked α-Xylp. The other branches might be linked by T-α-Glcp at C-6 of 1,4-linked α-Glcp. Based on the monosaccharide analysis, methylation, partial hydrolysis, and NMR analysis, a possible repeating unit for JCS1 was proposed as follows:

YJCS1 Induces Neurite Extension of PC-12 Cells
Considerable research has been carried out to investigate the antioxidant and immunomodulatory activity of acetyl derivatives of polysaccharides [24,28,29]. The acetylation (DS) of YJCS1 was 0.025 while the average molecular weight was 18.8 kDa.
Interestingly, we found that JCS1 did not extend the neurites of the PC-12 cells; however, the induction of the PC-12 cells was observed to be in a dose-dependent manner following treatment with the acetyl derivative of JCS1. The induced neuritogenesis was significant at low and high concentrations ( Figure 4F,H). The results suggested that this acetylated polysaccharide had the potential to be developed as a drug candidate to prevent or even treat neurodegeneration diseases.

YJCS1 Induces Neurite Extension of PC-12 Cells
Considerable research has been carried out to investigate the antioxidant and immunomodulatory activity of acetyl derivatives of polysaccharides [24,28,29]. The acetylation (DS) of YJCS1 was 0.025 while the average molecular weight was 18.8 kDa.
Interestingly, we found that JCS1 did not extend the neurites of the PC-12 cells; however, the induction of the PC-12 cells was observed to be in a dose-dependent manner following treatment with the acetyl derivative of JCS1. The induced neuritogenesis was significant at low and high concentrations ( Figure 4F,H). The results suggested that this acetylated polysaccharide had the potential to be developed as a drug candidate to prevent or even treat neurodegeneration diseases.

Discussion
In recent decades, differences have been discovered in the molecular weight, category, and molecular ratios of monosaccharide linkage types of polysaccharides extracted from Dendrobium plant species. However, there might be some common structural characteristics among Dendrobium plant polysaccharides. For example, 1,3-and 1,4-linked mannose residues are usually in the β configuration [1,23,30-32] while 1,2-and 1,6-linked mannose residues are in the α configuration
DCPP1a-1, isolated from the suspension-cultured protocorms of Dendrobium significantly inhibited hydroxyl radicals and superoxide anion radicals [34]. In another study [26], mice injected intraperitoneally with Dendrobium denneanum polysaccharide for 20 days showed lower serum malondialdehyde (MDA) and higher serum superoxide dismutase (SOD) than the control mice did. Some polysaccharides inhibit tumor cell growth in vitro [35] and in vivo [33,36]. The researchers considered the antitumor properties to be associated with the excellent immune-enhancing and antioxidant properties of the polysaccharide [33]. Moreover, considerable research has been focused on studying other bioactivities of dendrobium polysaccharides. For instance, the DCLP polysaccharide isolated from Dendrobium chrysotoxum had antidiabetic effects in alloxan-induced hyperglycemic mice [37]. In addition, the DHP-W2 polysaccharide from Dendrobium huoshanense might impede anti-glycation activity [27].
Currently, some acetylated polysaccharides from Dendrobium have been reported [1,2,23,30,31]. These polysaccharides were extracted from D. nobile, D. officinale, and D. huoshanense. The substitution position of their acetyl groups differed, and all the acetylated Dendrobium polysaccharides had bioactivities. It was observed that the acetyl groups were just substituted at the O-2 or O-3 of glucose and mannose [1,23,30,31]. Similarly, considerable research has shown that acemannan, an acetylated polymannose from Aloe vera, promoted tissue repair [38,39]. Furthermore, some investigations were carried out on the acemannan to determine the effect of the acetyl-groups on its physical and biological properties [40,41]. Previous studies have demonstrated the important role of acetyl groups on polysaccharide bioactivity [40]. The above research studies have provided two enlightening pieces of information or questions. One is that the bioactivities are different between the native active Dendrobium polysaccharides with acetyl groups and those without acetyl groups, which were deacetylated. Another important question is whether acetylated derivatives of novel Dendrobium polysaccharides, which mainly contain glucose or mannose have novel bioactivities. Therefore, in this study, the novel polysaccharide containing abundant glucose extracted from D. nobile was acetylated. Moreover, the acetylated polysaccharide, YJCS1, induced the neuritogenesis of PC-12 cells, which provides a reference for studying novel bioactivities of acetylated derivatives of Dendrobium polysaccharides.