Structure Identification of Two Polysaccharides from Morchella sextelata with Antioxidant Activity

Mushrooms of the Morchella genus exhibit a variety of biological activities. Two polysaccharides (MSP1-1, 389.0 kDa; MSP1-2, 23.4 kDa) were isolated from Morchella sextelata by subcritical water extraction and column chromatography fractionation. Methylation and nuclear magnetic resonance analysis determined MSP1-1 as a glucan with a backbone of (1→4)-α-D-glucan branched at O-6, and MSP1-2 as a galactomannan with coextracted α-glucan. Light scattering analysis and transmission electron microscopy revealed that MSP1-1 possessed a random coil chain and that MSP1-2 had a network chain. This is the first time that a network structure has been observed in a polysaccharide from M. sextelata. Despite the differences in their chemical structures and conformations, both MSP1-1 and MSP1-2 possessed good thermal stability and showed antioxidant activity. This study provides fundamental data on the structure–activity relationships of M. sextelata polysaccharides.


Introduction
Natural polysaccharides have attracted extensive attention because of their advantages, such as safety, biocompatibility, and in vivo biodegradability. Edible mushrooms are rich sources of natural polysaccharides. Mushroom polysaccharides have wide applications in the food industry as dietary supplements, nutritional promoters to flour and dairy products, or edible films for packaging materials [1].
Polysaccharides possess high capacities for carrying biological information because of their large structural variability. Chemically, most mushroom polysaccharides are glucans (α-, β-, or α/β) or heteropolysaccharides. However, polysaccharides with the same chemical structure can exhibit different biological activities after changes in conformation [2]. Information on the chemical composition, molecular weight (MW), primary structure (monosaccharide composition, skeleton structure, and branching structure), and advanced structure (three-dimensional chain conformation) of polysaccharides is important to understanding their activities. Therefore, the first step toward elucidating structure-activity relationships is to determine the molecular structure.
Ultraviolet-visible (UV-vis) spectroscopy was performed on the purified polysaccharides (1.0 mg/mL each) using a Shimadzu UV-2550 spectrophotometer (Shimadzu, Japan). The scan-related parameters were set as scan interval 0.5 nm and scan range 200-400 nm.
Constituent sugar analysis of the polysaccharide fractions was conducted using an ion chromatography system (ICS-5000) equipped with a DionexCarbopac TM PA20 (150 mm × 3.0 mm id) column and pulsed amperometric detector (PAD). Each sample (10 mg) was precisely weighed and hydrolyzed for 3 h with trifluoroacetic acid (3 M, 10 mL) at 120 • C. The acid hydrolysate was dried under nitrogen and mixed thoroughly with 5 mL distilled water. The mixture (100 µL) was then drawn into deionized water (900 µL) and centrifuged. The supernatant (5 µL) was analyzed by ICS-5000 and eluted with a mobile phase (H 2 O, 15 mM NaOH, and 100 mM sodium acetate solution) at 30 • C and a set elution flow rate of 0.3 mL/min.

Methylation
The samples were methylated using the method described previously [12]. MSP1-1 and MSP1-2 were permethylated according to the following steps: first, 10 mg of MSP1-1 or MSP1-2 was weighed and dissolved in 500 µL of DMSO, and 50 µL of DMSO/NaOH solution (120 mg/mL) was added to the sample solution to initiate a reaction. After 30 min, iodomethane (50 µL) was added and reacted for 1 h. The reaction was terminated by the addition of deionized water (1 mL). The product was subsequently extracted using dichloromethane (500 µL) and dried with nitrogen. Trifluoroacetic acid (2 M) was added to the product after methylation was completed, and a hydrolysis procedure was performed (reaction at 121 • C for 90 min). Finally, the obtained product was reduced with 2 M ammonia and 1 M NaBD 4, and acetylated with acetic anhydride. To detect the ultimate derivatives, an Agilent 7890A-5977B gas chromatography-mass spectrometry (GC-MS) instrument (Santa Clara, CA, USA) with a BPX70 (25 m × 0.22 mm × 0.25 µm, Trajan) was employed with high-purity helium as carrier gas at an injection volume of 1 µL. The temperature of the BPX70 column was initially 140 • C (held for 2.0 min) and then ramped up to 230 • C (held for 3 min) using a 3 • C/min program. The peaks of the derivatives were identified from the relative retention times and mass spectral data.

Nuclear Magnetic Resonance (NMR) Spectroscopy
The NMR data were obtained using a 500 MHz Bruker NMR spectrometer (Zurich, Switzerland) at 25 • C. MSP1-1 and MSP1-2 (20 mg each) were dissolved in D 2 O solvent and lyophilized. The dried polysaccharides (20 mg) were transferred to a standard 5 mm probe after dissolution in 500 µL D 2 O solvent. The operating frequencies of 1 H NMR and 13 C NMR were 500 and 125 MHz, respectively. The acquisition times were set to 16 times for 1 H NMR spectra, 1024 times for 13 C NMR spectra, 12 times for 1 H/ 1 H correlation spectroscopy (COSY), 12 times for heteronuclear single quantum correlated spectroscopy (HSQC), 32 times for nuclear overhauser effect spectroscopy (NOESY), and 64 times for heteronuclear multiple bond correlation (HMBC) spectra. MestReNova software (Version: 9.1.0-14011; Mestrelab Reserch, Santiago de Compostela, Spain) was used to process the NMR data of the polysaccharide samples.

Congo Red Test
The presence of helical structures in the polysaccharide samples was tested using a method described by Jia et al. [13]. That is, the sample solution (1.0 mg/mL, 1.0 mL), Congo red (80 mM, 1.0 mL), and NaOH (1.0 M) were mixed until the NaOH concentration in the mixed solution gradually increased to 0.5 M. The blank control was a Congo red solution without polysaccharides. The UV-2550 spectrometer was run to scan the maximum absorption wavelength (λ max ) at each NaOH concentration.

Light Scattering Measurements and Morphological Observations
The polysaccharide samples were dissolved in 0.9% NaCl solution (3 mg/mL) for conducting dynamic light scattering (DLS) measurements using a Brookhaven light scattering spectrometer (BI-200SM, Brookhaven, GA, USA) at a wavelength of 532 nm and a 90 • angle. The sample solutions were diluted to varying concentrations (0.1-0.75 mg/mL) by using 0.9% NaCl solution and stirred for 24 h after each dilution. The solutions were measured at angles of 50 • -140 • for static light scattering (SLS). All solutions were purified (filter: 0.22 µm) three times before testing. The obtained data were fitted using Berry plots.
The chain conformation of the polysaccharides was assessed using a Tecnai G2 Spirit Bio transmission electron microscope (TEM; FEI, Portland, OR, USA) at 80 kV. MSP1-1 or MSP1-2 was dissolved in 0.9% NaCl solution to achieve a final concentration of 5 µg/mL. A drop of the polysaccharide solution was deposited on the support carbon film (200 mesh), dried in the air, and stained with 0.2% phosphotungstic acid.

Thermal Analysis
Simultaneous thermogravimetric and differential scanning calorimetry (TG-DSC) was performed using an STA 449 F3 thermogravimetric analyzer (Netzsch, Selb, Germany). The samples (5 mg each) were sealed in an Al 2 O 3 crucible and heated from 25 • C to 600 • C (heating rate: 10 • C/min) under a nitrogen atmosphere.

Antioxidant Activity
The antioxidant activity (DPPH radical scavenging and ferric reducing activity) test of the polysaccharides was measured as described previously [11]. The specific procedures are supplied in supplementary data.

Statistical Analysis
All data were obtained using three independent trials, and results were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed using SPSS 18.0 software. The Tukey test results were significantly different in cases where the p-values were less than 0.05.

Thermal Analysis
Simultaneous thermogravimetric and differential scanning calorimetry (TG-DSC) was performed using an STA 449 F3 thermogravimetric analyzer (Netzsch, Selb, Germany). The samples (5 mg each) were sealed in an Al2O3 crucible and heated from 25 °C to 600 °C (heating rate: 10 °C/min) under a nitrogen atmosphere.

Antioxidant Activity
The antioxidant activity (DPPH radical scavenging and ferric reducing activity) test of the polysaccharides was measured as described previously [11]. The specific procedures are supplied in supplementary data.

Statistical Analysis
All data were obtained using three independent trials, and results were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed using SPSS 18.0 software. The Tukey test results were significantly different in cases where the p-values were less than 0.05.

Methylation Analysis of MSP1-1 and MSP1-2
The GC-MS results for the methylation analysis are shown in Table 1

Methylation Analysis of MSP1-1 and MSP1-2
The GC-MS results for the methylation analysis are shown in Table 1 and Figure S3

NMR Analysis of MSP1-1 and MSP1-2
Detailed structural information regarding MSP1-1 and MSP1-2 was elucidated based on the chemical shifts in the NMR spectra. The 1 H NMR spectrum of MSP1-1 ( Figure 3A) reveals a large number of resonance signals of overlapping protons in the δ 3.0-5.5 ppm region. The signals appear in the anomeric region at δ 5.35, δ 5.31, δ 4.92, δ 4.92, δ 5.18, and δ 4.58 ppm, and are denoted by A, B, C, D, Rα, and Rβ, respectively. In the 13 C spectrum of MSP1-1 ( Figure 3B), the anomeric carbon signals of residues A, B, C, D, Rα, and Rβ were determined to be at δ 100.0, δ 100.0, δ 98.5, δ 99.9, δ 92.0, and δ 95.8 ppm, respectively.  After the origin of the anomeric signal was determined, the NMR spectra and methylation results were compared with the reported data for similar glycosyl substitutions [5,19]. All chemical shifts were assigned to the residues of MSP1-1 and given in Table 2. The HMBC spectrum ( Figure 3E) reveals the following correlations: A H-1/A C-4, A H-1/B C-4, and A H-4/A C-1. The linkages of the polysaccharide samples are further verified by the NOESY spectrum ( Figure 3F). Results of the methylation and NMR analyses indicate that MSP1-1 is composed of residue A through a 1→4 glycosidic bond, and that residues A and B are connected to form the sugar backbone. On the branch, residues B and C are connected to residue D through position 6. The possible repeating structural units of MSP1-1 are shown in Figure 3G.

Triple Helix Structural Determination
Congo red solution is an indicator for identifying the triple helix conformations in polysaccharides [30]. With an increasing concentration of NaOH, no characteristic red shift was observed at λ max related to the polysaccharide-Congo red complexes (shown in supplementary date, Figure S4), indicating that neither MSP1-1 nor MSP1-2 had a triple helix structure.

Conformational Characteristics
Polysaccharides tend to aggregate in water, and the chain conformation in 0.9% NaCl solution was studied by light scattering (shown in supplementary date, Figure S5). MSP1-1 showed a single particle size distribution with a hydrodynamic radius (R h ) of 28.89 nm. MSP1-2 aggregated in 0.9% NaCl solution and yielded a bimodal size distribution with a R h of 34.87 nm. The calculated values of the conformational parameters are presented in Table 3. The second virial coefficient (A 2 ) of MSP1-1 is positive, indicating that 0.9% NaCl solution is a good solvent, whereas A 2 is negative for MSP1-2, showing that aggregates existed in MSP1-2/0.9% NaCl solution [31]. Both MSP1-1 and MSP1-2 had a random coil conformation as their structure parameter ρ = R g /R h values (R g : radius of gyration) were calculated as 1.89 and 1.46, respectively [32]. The chain conformations of MSP1-1 and MSP1-2 were verified using TEM in 0.9% NaCl solution. In Figure 5A, MSP1-1 resembles a random coil chain, which is consistent with the light scattering results. MSP1-2 exhibits a unique fishnet-like conformation ( Figure 5B). Similar aggregates have been reported in lentinan polysaccharides, and the driving force for this aggregation was hydrogen bonding [33]. This is the first time that a network structure has been observed in an M. sextelata polysaccharide. In this conformation, the polysaccharide may exhibit utility as a carrier.

Thermal Properties
TGA curves indicate two-stage decomposition for both MSP1-1 and MSP1-2 ( Figure S6). The first stage of degradation occurred before 100 • C due to the rapid evaporation of free water [34] with a weight loss of 10.49% for MSP1-1 and 8.89% for MSP1-2. The second degradation occurred in the temperature interval of 140-400 • C, and the weight loss of MSP1-1 and MSP1-2 were 74.56% and 82.49%, respectively. This is related to the thermal decomposition of the polysaccharides [35]. The derivative thermogravimetric curves revealed that the maximal degradation of MSP1-1 and MSP1-2 occurred at 308.50 • C and 309.01 • C, respectively. This thermal behavior was similar to that observed for polysaccharides derived from Ribes nigrum L. [36] and Ocimum album L. seed [37].

Thermal Properties
TGA curves indicate two-stage decomposition for both MSP1-1 and MSP1-2 ( Figure  S6). The first stage of degradation occurred before 100 °C due to the rapid evaporation of free water [34] with a weight loss of 10.49% for MSP1-1 and 8.89% for MSP1-2. The second degradation occurred in the temperature interval of 140-400 °C, and the weight loss of MSP1-1 and MSP1-2 were 74.56% and 82.49%, respectively. This is related to the thermal decomposition of the polysaccharides [35]. The derivative thermogravimetric curves revealed that the maximal degradation of MSP1-1 and MSP1-2 occurred at 308.50 °C and 309.01 °C, respectively. This thermal behavior was similar to that observed for polysaccharides derived from Ribes nigrum L. [36] and Ocimum album L. seed [37].
The DSC thermograms of MSP1-1 and MSP1-2 were consistent with the TGA results, revealing two marked endothermic transitions attributed predominantly to the dehydration and thermal decomposition of the polysaccharides. Degradation began at 261.70 °C for MSP1-1 with a maximal peak at 305.06 °C, and at 279.69 °C for MSP1-2 with a maximal peak at 315.57 °C. The higher decomposition temperature of MSP1-2 indicates that it has better thermal stability compared to MSP1-1 [38]. Additionally, the higher endothermic enthalpy change (ΔH = 263.86 J/g) confirms that MSP1-2 possesses better thermal stability [39], which may be related to its shorter chain.

Antioxidant Activity In Vitro
Compared to the positive control group (Vc), MSP1-1 and MSP1-2 can scavenge free DPPH radicals ( Figure 6A). The scavenging activity of samples was correlated with increasing concentration (0.5-4 mg/mL). At a concentration of 4 mg/mL, the scavenging activities of MSP1-1 and MSP1-2 were 43.41% and 58.09%, respectively, suggesting that MSP1-2 has higher antioxidant activity than MSP1-1 (p < 0.05). This activity is related to the chemical structure of MSP1-2 because polysaccharides containing glucose and mannose were reported to have good antioxidant activity. Moreover, the higher molecular weight of MSP1-1 affects its scavenging ability [12,40]. The scavenging activity of pure M. sextelata polysaccharide was significantly decreased compared with that of crude polysaccharides at the corresponding concentration (p < 0.05) [11] because some active substances with antioxidant properties have been removed in the purification process. The ferric reducing abilities of MSP1-1 and MSP1-2 were significantly decreased compared to that of the Vc group (p < 0.05). However, they still showed a certain reducing ability, which was higher than that of Laminaria japonica polysaccharides at 2.0 mg/mL [41]. In summary, these results showed that M. sextelata polysaccharides have antioxidant The DSC thermograms of MSP1-1 and MSP1-2 were consistent with the TGA results, revealing two marked endothermic transitions attributed predominantly to the dehydration and thermal decomposition of the polysaccharides. Degradation began at 261.70 • C for MSP1-1 with a maximal peak at 305.06 • C, and at 279.69 • C for MSP1-2 with a maximal peak at 315.57 • C. The higher decomposition temperature of MSP1-2 indicates that it has better thermal stability compared to MSP1-1 [38]. Additionally, the higher endothermic enthalpy change (∆H = 263.86 J/g) confirms that MSP1-2 possesses better thermal stability [39], which may be related to its shorter chain.

Antioxidant Activity In Vitro
Compared to the positive control group (Vc), MSP1-1 and MSP1-2 can scavenge free DPPH radicals ( Figure 6A). The scavenging activity of samples was correlated with increasing concentration (0.5-4 mg/mL). At a concentration of 4 mg/mL, the scavenging activities of MSP1-1 and MSP1-2 were 43.41% and 58.09%, respectively, suggesting that MSP1-2 has higher antioxidant activity than MSP1-1 (p < 0.05). This activity is related to the chemical structure of MSP1-2 because polysaccharides containing glucose and mannose were reported to have good antioxidant activity. Moreover, the higher molecular weight of MSP1-1 affects its scavenging ability [12,40]. The scavenging activity of pure M. sextelata polysaccharide was significantly decreased compared with that of crude polysaccharides at the corresponding concentration (p < 0.05) [11] because some active substances with antioxidant properties have been removed in the purification process. The ferric reducing abilities of MSP1-1 and MSP1-2 were significantly decreased compared to that of the Vc group (p < 0.05). However, they still showed a certain reducing ability, which was higher than that of Laminaria japonica polysaccharides at 2.0 mg/mL [41]. In summary, these results showed that M. sextelata polysaccharides have antioxidant capacity and may be potentially used as a natural antioxidant.

Conclusions
In the present study, we successfully isolated two novel polysaccharides (MSP1-1 and MSP1-2) via the subcritical water extraction from M. sextelata fruiting bodies. The structure, chain conformation, and thermal stability of these two polysaccharides were investigated using different analytical methods. MSP1-1 displayed random coil chains with a backbone of α-1, 4-linked Glcp, which was substituted at the O-6 by a short branch. MSP1-2 showed a network chain with two domains, a backbone of α-1, 2-linked Manp and α-1, 6-linked Manp, and another α-1, 4-linked Glcp backbone. Both polysaccharides showed antioxidant activity with some variations due to differences in their chemical structures and molecular weights. This research outcome will be beneficial to the development of green applications in the food and medical sciences. The antioxidant activity in vivo of M. sextelata polysaccharides requires additional investigations.

Conclusions
In the present study, we successfully isolated two novel polysaccharides (MSP1-1 and MSP1-2) via the subcritical water extraction from M. sextelata fruiting bodies. The structure, chain conformation, and thermal stability of these two polysaccharides were investigated using different analytical methods. MSP1-1 displayed random coil chains with a backbone of α-1, 4-linked Glcp, which was substituted at the O-6 by a short branch. MSP1-2 showed a network chain with two domains, a backbone of α-1, 2-linked Manp and α-1, 6-linked Manp, and another α-1, 4-linked Glcp backbone. Both polysaccharides showed antioxidant activity with some variations due to differences in their chemical structures and molecular weights. This research outcome will be beneficial to the development of green applications in the food and medical sciences. The antioxidant activity in vivo of M. sextelata polysaccharides requires additional investigations.