Wild plants of D. huoshanense growing in Huoshan County, Anhui Province, China, were collected. Dimethyl sulfoxide (DMSO), acetonitrile, sodium hydride (NaH), and iodomethane (CH₃I) lipopolysaccharide (LPS) were purchased from Sigma Chemical Co., USA. DEAE-cellulose and Sephadex G-100 were purchased from Solarbo Co., Ltd. (Beijing, China). The Cell counting kit-8 (CCk-8) was purchased from Han Heng Biotechnology Co., Ltd. (Shanghai, China). The ascorbic acid (VC) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the other reagents or chemicals were of analytical grade.

The stems were air-dried, ground to powder and refluxed with 95% ethanol (1:200, g/mL) for three times, each time for 5 h. This process was done to remove the small molecular compounds. A Synthware® pressure glass vial (Beijing Synthware Glass, Inc., China) with a volume of 350 mL was used for the D. huoshanense polysaccharide extraction by HCW, which is water under subcritical pressure and temperature. The air-dried powder of D. huoshanense (5 g) and water (100 mL) were added into the reactor, and the reactor was sealed. The mixture was heated up to 150°C (0.4757 Mpa). The sample was extracted for 180 min and each sample was extracted in this way two times. After centrifugation, the supernatants from the two extractions were combined and concentrated, and then precipitated by adding 4 times the volume of anhydrous alcohol. The precipitate was collected by centrifugation, redissolved in water and deproteinated by the Sevage method (polysaccharide solutions:chloroform:isopentanol = 25:4:1, v/v/v) 6 times. The deproteinized polysaccharides were then decolorized by HP20 macroporous resin at 25°C for 4 h (120 r/min). The supernatant was combined, concentrated and dialyzed using 3,500 Da, and finally lyophilized. The yield of crude polysaccharides was calculated by the following equation:

The crude polysaccharide was further purified using DEAE-cellulose and Sephadex G-100. Then, the eluents were collected and determined using the phenolsulfuric acid method using α-glucose as a standard, and elution curves were drawn. Finally, white (DHPs-1) purified polysaccharides was obtained. The yields of DHPs-1 were calculated by the following equation:

The total carbohydrate content, uronic acid content and protein content was determined by phenol-sulfuric acid method using D-glucose as a standard, mhydroxydiphenyl method and using galacturonic acid as a standard and Coomassie brilliant blue method with bovine serum albumin as a standard, respectively (8).

The antioxidant activities of DHPs-1 had been determined using Vc as the positive control, and the macrophage proliferation and Q-PCR assays were determined using LPS as positive controls.

All experiments were tested in triplicate. The data were analyzed using IBM SPSS Statistics 20.0, and mean comparisons were carried out using Duncan's multiple range test. A value of p < 0.05 was considered to be statistically significant.

Dendrobium huoshanense polysaccharide (DHP) was obtained using subcritical water extraction with a yield of 19.52%, which is the ratio of the crude polysaccharide to the raw material. The crude polysaccharide was deproteined and decolourized, and then further purified by the DEAE-52 column and Sephadex G-100 chromatography column. As shown in Figure 1, purification on the DEAE-52 column yielded four fractions: one deionized water fraction, two 0.1 moL/L NaCl fractions, and one 0.3 moL/L NaCl fraction. Their yields were: 21.5, 1.9, 6.0, and 2.4%, respectively. Due to the high yield of the deionized water fraction, it was further purified using a Sephadex G-100 chromatography column, and yielded a single fraction with a symmetrical peak with a yield of 89.3% (not shown); this fraction was named DHPs-1. The calculated weight-average molecular weight (Mw) and number-average molecular weight (Mn) of DHPs-1 were 50 and 36 KDa, respectively (Table 2). The polymer dispersity index (PDI) of DHPs-1 was about 1.39; the closeness to 1 suggests DHPs-1 was homogeneous. This result is different from the polysaccharides obtained from Dendrobium huoshanense (6.7 KDa, 232 KDa-8090 KDa) using the traditional extraction method (11–14). XRD analysis is often used to detect the crystalline or amorphous structure of the polysaccharides. The XRD image of DHPs-1 showed a strong diffraction peak at 2θ of 20°, and this result indicates that there was not only crystalline structure but also amorphous structure in DHPs-1 (see Supplementary Material). In fact, DHPs-1 had more amorphous than crystalline with the crystallinity degree of 18.5%. Actually, this crystal structure is directly determined by the flexibility, solubility, swelling and other properties of polysaccharides (15). This result suggests the DHPs-1 is a novel polysaccharide, and the reason was attributed to the different species, specific extraction and processing methods.

The FT-IR spectrum of DHPs-1 showed two characteristic peaks: a strong one at around 3,423 cm⁻¹ representing the intense stretching vibration of O-H bonds, and a weak one at around 2,923 cm⁻¹ representing weak C-H bonds (Figure 1B). The areas at 1,734 and 1,636 cm⁻¹ indicated the presence of -COOR and -COOH, respectively, which indicates the existence of galacturonic acid or glucuronic acid and acetyl groups (15). The band between 950 and 1,200 cm⁻¹ is often called the “fingerprint” of molecules, because the position and intensity of the bands are specific for different polysaccharide. The bands at 1,636 and 1,249 cm⁻¹ revealed the presence of little amount of protein, which is consistent with above result (16). This result is in accordance with the result of the composition analysis shown in Table 1. The signals at 1,200–1,000 cm⁻¹ indicated that DHPs-1 contained pyranose rings, which was attributed to C-O-C and C-O-H glycosidic bands (17). The band at 1,063 cm⁻¹ indicated that the glucosidic linkages of the glucosyl residues were β-linked due to O-substituted glucose residues (18). In addition, the peaks at 890 and 811 cm⁻¹ might be due to the presence of manopyranosyl and α-galactopyranosyl. In addition, the peak at 890–900 cm⁻¹ was indicative of the presence of β-glycosidic linkages and the absorption peak at 853 cm⁻¹ indicated the presence of α-glycoside bonds (7).

DHPs-1 is mainly composed of glucose, mannose, galactose, galacturonic acid, rhamnose with the ratio of 65.04:14.23:8.17:6.41:2.34 (Table 3). There are also traces of xylose, arabinose, glucuronic acid, fructose, and D-glucosamine hydrochloride with the ratio of 1.25:0.95:0.61:0.24:0.24. Other studies have reported the component monosaccharides of D. huoshanense polysaccharides as follows: DHPD2 was mainly composed of galactose, glucose and arabinose with a molar ratio of 0.90:0.72:0.20 and traces of mannose, rhamnose and xylose (12); DHPIA was mainly composed of mannose, glucose and galactose with a molar ratio of 2.5:16.0:1.0 (11); TC-DHPA4 was composed of arabinose, rhamnose, mannose, glucose, galactose and glucuronic acid in a molar ratio of 1.00:1.28:1.67:4.71:10.43:1.42 (14); cDHPS was composed of d-mannose and d-glucose in a 3.04:1.00 molar ratio (13); DHP-4A was composed of glucose, mannose, arabinose and rhamnose in a molar ratio of 13.80:6.10:3.00:2.10 (19); HPS-1B23 was composed of glucose, mannose and galactose in a 31.00:10.00:8.00 molar ratio (20). The differences of molar ratio and the monosaccharide types are presumably due to different source material, and different extraction and purification methods.

Methylation is a powerful tool to detect the polysaccharide's structure. Due to the low content of uronic acid, all the free hydroxyl groups of DHPs-1 are directly and completely methylated. The methylated DHPs-1 was hydrolysed, reduced and acetylated to obtain the PMAAs, and then were detected by GC-MS. The GC-MS result indicated that DHPs-1 has seven types of sugar linkages: Manp-(1→, → 4)-Manp-(1→, → 4)-Galp-(1→, → 4)-Glcp-(1→, → 3, 4)-Galp-(1→, → 4, 6)-Galp-(1→, → 3, 4, 6)-Galp-(1→ at molar percent ratios of 12.1:2.1:1.3:69.3:4.8:9.3:1.1, respectively (Table 4). Both results of monosaccharide composition and methylation linkage analysis, glucose was the main component of DHPs-1, which indicated that 1,4-Glcp residues exist in the backbone of DHPs-1. These molar percent ratios of different types of sugar residues were consistent with the percentages of sugars obtained above, except for galactose. This reason could be that the galacturonic acid peak overlapped with the galactose peak, causing an apparent increase of galactose content in the methylation result. The linkages of galacturonic acid could be the 3,4-GalpA or 4,6-GalpA. The number of terminal Manp residues approximately equaled the number of branched residues, indicating that the methylation method of DHPs-1 was effective. The terminal mannose was directly or indirectly attached to → 3, 4)-Galp-(1→, → 4, 6)-Galp-(1→, and → 3, 4, 6)-Galp-(1→, as the ratio between them was close to 1:1.

The DHPs-1 structure was further determined by 1D NMR and 2D NMR. The ¹H, ¹³C, DEPT-135, HSQC, HMBC COSY ROESY spectra of DHPs-1 are shown in Figure 2. Three anomeric signals were observed in the region of 4.40–5.40 ppm for the ¹H NMR (Figure 2A), suggesting that DHPs-1 was mainly composed of three sugar residues, and the glycosidic linkages contain β- and α-type. In the ¹³C NMR spectrum (Figure 2B), three anomeric carbon signals were observed at 100.08, 99.70, and 102.4 ppm, and the remaining signals appeared in the region of 60.21–78.42 ppm. The three sugar residues were designated as A, B, and C, respectively. According to the HSQC spectrum (Figure 2C), the anomeric carbon signals at A (δ 100.08), B (δ 99.70), and C (102.40 ppm) correlated to anomeric proton signals of δ 4.63, 5.27, and 4.39 ppm, respectively. The chemical shifts in the ¹³C spectrum and the DEPT-135 spectrum were similar. However, the CH₃ and CH signals were positive, and the CH₂ signals were negative in the DEPT-135 spectrum. According to the DEPT-135 spectrum (see Supplementary Material), C-6 signal peaks were mainly observed in the region of δ 60–70 ppm. Three main C-6 signal peaks, including δ 60.50, 62.36, and 60.21 ppm, were obtained (21). The anomeric signal (100.02 ppm) of 1,4-β-D-Manp was overlapped with the signal of 1,4-α-D-Glcp. No signal existed in the region of 82.0–88.0 ppm, indicating that all the sugar residues were of pyranose configuration. According to the literatures, 19.0–20.7 ppm and 172.6–174.1 ppm were investigated to be Me and CO of O-acetyl groups. This was also supported by the chemical shifts existed at 2.00–2.07 ppm in the ¹H NMR spectrum of DPHs-1 (20). This result is in accordance with the results of monosaccharide composition and FT-IR analysis.

According to the COSY spectrum (Figure 2D), the chemical shifts observed at 4.05, 3.61 3.54, 3.66, and 3.56 ppm were assigned to H2, H3, H4, H5, and H6 of residue A, respectively. Taking into consideration of the ¹³C NMR and HSQC spectra, the corresponding ¹³C shifts can be obtained at 72.92, 78.24, 72.71, 76.15, and 60.50 ppm. Above all, residue A was the 1,4-β-d-Glcp (21, 22). The similar way can be used to assign the proton and carbon signals for residue B and residue C in 1D and 2D NMR spectra. Thus, the residues were designated as terminal-α-Manp (B), and 1,4,6-β-D-Galp (C), respectively. The main chemical shifts of DHPs-1 are presented in Table 5.

The sequence of sugar residues was determined using HMBC, and the backbone structure of DHPs-1 was analyzed using HMBC spectrum. In the HMBC spectrum (Figure 2E), the chemical signals of DHPs-1 were assigned by a combination of the 1D and 2D NMR spectra. For example, the cross signal at 3.54/100.08 ppm revealed that the H-4 of → 4)- β-D-Glcp-(1→ and the C-1 of → 4)-β-D-Glcp-(1→ were connected, indicating the presence of → 4)-β-D-Glcp-(1→ 4)-β-D-Glcp-(1→. The cross signal at 4.63/69.84 ppm revealed that the H-1 of → 4)-β-D-Glcp-(1→ and the C-4 of → 4, 6)-β-D-Galp-(1 were connected, indicating the presence of → 4)-β-D-Glcp-(1→ 4, 6)-β-D-Galp-(1→. The cross signal at 4.63/76.5 ppm revealed that the H-1 of → 4)-β-D-Glcp-(1→ and the C-4 of α-D-Manp-(1 were connected, suggesting the presence of α-D-Manp-(1→ 4)-β-D-Glcp-(1→ (23–25). The ROESY spectrum of DPHs-1 (Figure 2F) shows cross-peaks: H1 of 1,4-β-D-Glcp with H2, H3, H5 at 4.63/4.05, 3.58, and 3.66 ppm, respectively; H1 of terminal-Manp with H4 at 5.27/3.84 ppm, revealing the presence of sugar residues: … → 4)-β-D-Glcp-(1→ 4)-β-D-Glcp-(1→ … and α-D-Manp → 4)-β-D-Glcp-(1→.

SEM was used to characterize the morphology of DHPs-1. Under high magnification (800× and 1,000×), it appeared as fibrous filaments and ribbons with branches (Figure 3). These microstructural characteristics may be attributed to the structure disruption of the cell wall and the disaggregation of the polysaccharide aggregates by the subcritical water extraction process, leading to the loosened texture of DHPs-1. The morphology of DHPs-1 was mainly affected by the subcritical water extraction method and the purification step, which could change the hydrogen bonds of polysaccharide molecule. The surface morphology and microstructure of DHPs-1 might be correlated with its physicochemical properties and antioxidant activities (26).

Changes in thermal stability can quantitatively reflect changes of material quality in the process of dehydration, oxidation, and decomposition. The thermal property of DHPs-1 was investigated using TG, DTG, and DSC analyses. Two stages of weight losses were obtained from the TG experiment over a wide temperature range (25–760°C) (Figure 4). The first weight loss occurred below 150°C with 4.70% of the original weight, which might be attributed to the loss of bounding water. The second stage of weight loss was observed at 200–350°C. The weight of DHPs-1 began to decrease sharply, and this could be attribute to the depolymerization and decomposition of DHPs-1. The weight loss in this region was 78.86% of the original weight. The maximum decomposition rate of DHPs-1 seen in the DTG curve occurred at 316.43°C. Simultaneously, the DSC curve was obtained. As shown in Figure 4, the DHPs-1 was characterized by one peak at 87.6°C corresponding to the glass transition temperature. One exothermic peak at 200–350°C was detected, which might be attributed to the chemical decomposition of DHPs-1 (27). Taking into consideration of TG, DTG, and DSC curves, DHPs-1 was relatively stable below 300°C. This result indicated that the thermal stability of DHPs-1 was higher than that of polysaccharides from Folium Isatidis (25). Consequently, there are differences in their thermal stabilities and these differences may be attributed to the different source material, extraction method, purification method, spatial conformation, monosaccharide compositions, or structures of polysaccharides.

The DPPH· and ·OH scavenging activities of DHPs-1 are presented in Figure 5. The scavenging activity of DHPs-1 increased with the increase of polysaccharide concentration (0.05–10 mg/mL) (Figure 5A). At a concentration of 10 mg/mL, the DPPH· scavenging activity was 92.64%, which was close to that of Vc. The Dendrobium huoshanense polysaccharide was extracted twice with distilled water at 65°C and its scavenging rates of DPPH were increased from 31.4 to 38.7% with increasing the polysaccharide concentration from 0.5 to 2.0 mg/mL (11). The DPPH· scavenging activity of DHPs-1 was better than that of Dendrobium huoshanense polysaccharide obtained by traditional water extraction method. This might be attributed the different monosaccharide composition, molecular weight, chain conformation of polysaccharides obtained by different extracted and purified methods. The scavenging activity for hydroxyl radicals of DHPs-1 was in a concentration-dependent manner. The hydroxyl radical scavenging capacity of DHPs-1 (0.05–10 mg/mL) increased from 5.06 to 19.76% at concentrations between 0.05 and 10 mg/mL. With this level of antioxidant activity, DHPs-1 could be developed and used in natural medicine and functional food production (28). However, the antioxidant mechanism of DHPs-1 is unclear at present, and need to be investigated further.