Structural Characterization and Immunomodulatory Activity of Polysaccharides from Polygonatum sibiricum Prepared with Deep Eutectic Solvents

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Introduction
Polysaccharides are commonly extracted by using the pharmacopoeia or the acid (alkali) extraction methods, which are assisted by ultrasound, microwaves, and enzymes.However, there are some inherent limitations in these methods, such as high energy consumption, long extraction time, low extraction efciency, and poor selectivity [1].Traditional solvents (hot water, acid, and alkali) extract functional components in natural plants though destroying cell walls.However, ionic liquids can dissolve plant cell walls to release functional components from cells, so as to achieve the goal of improving the extraction ratio.Deep eutectic solvents (DES) is also considered to be ionic liquids, which has more excellent properties than other ionic liquids, such as lower cost, easy to prepare and store, inexpensive, biodegradability, and safety, as well as sustainability [2,3].DES is a mixture of hydrogen acceptor and hydrogen donor heated and dissolved proportionately, which remains uniform and transparent at room temperature [4].Te most widespread hydrogen acceptor is choline chloride (CC), yet the selection of hydrogen donors is diversifed, including oxalic acid, glucose, sucrose, lactic acid, ethylene glycol etc. [5,6].Among them, oxalic acid is relatively safe in food processing.Meanwhile, studies showed that the yield of polysaccharides using DES (CC/oxalic acid) as a solvent was higher than that of using other types of DES, which might be due to its stronger hydrogen bonding ability and electrostatic interactions with polysaccharides [7,8].At present, DES has been successfully used to extract natural product, such as polysaccharides [9], favonoids [10], proteins [11], and nicotinic acid [12].
Polygonatum sibiricum, also known as Jitou ginger, is widely cultivated in China [13].In Traditional Chinese Medicine, P. sibiricum is known for improving vital energy, nourishing the heart and lungs, and strengthening the muscles and bones [14].Studies showed that P. sibiricum mainly contained polysaccharides, saponins, favonoids, and other chemical components, while polysaccharides were one of the main active compounds [3,15].Polygonatum sibiricum polysaccharides perform antioxidant, antiaging, and immunomodulatory activity, and they also regulate glucose and lipid metabolism [16][17][18][19].
Polysaccharides can regulate immunomodulatory activity by enhancing the function of macrophages [20].Wu found that polysaccharides from Guapian tea leaves (LGP-1) could signifcantly stimulate the phagocytosis of macrophages, promote NO generation, and trigger TNF-α, IL-6, and IL-1β release dose-dependently.LGP-1 enhanced macrophage immunity via PI3K/AKT and NF-κB signaling pathways, and TLR4 was involved in the process [21].Polygala tenuifolia willd.polysaccharides could promote cell proliferation, increase the secretions of TNF-α, IL-6, and IL-1β, upregulate the expressions of P-p38, P-ERK, and P-JNK, activate macrophages, and exert immune activity by activating MAPKs mediated pathway [22].Tis study analyzed the structural characterization of P. sibiricum polysaccharides and investigated their immunomodulatory activity in RAW 264.7 cells.(Shanghai, China).All other chemicals and reagents used were of analytical grade.

Preparation and Purifcation of Polysaccharides.
Te dried rhizome of P. sibiricum was sliced and crushed with a pulverizer (BJ-800A; Deqing Baijie Electric Appliance Co., Ltd, Huzhou, China), which was then passed through a 0.42 mm (60 mesh) sieve to obtain the dried P. sibiricum powder.In Figure 1, the powder was immersed in 80% ethanol at room temperature with the ratio of 1 : 50 (m/v) for 30 min.Te solution was centrifuged at 6,000 rpm for 10 min to remove lipophilic compounds, and the precipitate was collected for subsequent experience.
Te pharmacopoeia method (National Pharmacopoeia Commission, 2020) was used as a comparison.In brief, the obtained precipitate was extracted with deionized water (1 : 50, g/mL) at 100 °C for 2 h.Te supernatant was combined and concentrated using a rotary evaporator (RE-52AA, Shanghai Yarong Biochemical Instrument Factory, Shanghai, China), the concentrated solution precipitated with 80% ethanol and stored at 4 °C for 12 h, and then the obtained precipitate was collected by centrifugation and freeze-dried.Subsequently, the protein was removed by the Sevag reagent (chloroform:n-butanol � 4 : 1, v/v), and the pigment was removed by the AB-8 macroporous resin column (3.5 cm × 20 cm).Finally, the product was lyophilized to obtain the crude polysaccharides PSP.
In the DES extraction method [20], 1 g of obtained precipitate was extracted with 20 mL of DES (choline chloride: oxalic acid = 1 : 1, mass ratio) at 70 °C for 40 min.After centrifugation (7,000 rpm, 8 min), the obtained supernatant was collected, precipitated with alcohol, deproteinized, and removed pigments, as stated by the pharmacopoeia method above.Te obtained polysaccharides were named as DPSP.DPSP was dissolved in deionized water (5 mg/mL).Te solution was loaded onto a DEAE-Sepharose Fast Flow column (3.5 cm × 20 cm), and the column was sequentially eluted with ultrapure water and 0.1, 0.2, 0.4, and 0.6 M sodium chloride (NaCl) solution at a fow rate of 1.0 mL/min.Te eluted fractions were collected (5 mL/tube) and monitored.Ten, the eluted fraction (0.04 M NaCl aqueous solutions) was concentrated, dialyzed with deionized water for 48 h at 4 °C, and freeze-dried to obtain the purifed polysaccharides DPSP-3.DPSP-3 (5 mg/ mL) was further purifed with Sephadex G-200 column (3.5 cm × 60 cm).Briefy, DPSP-3 was dissolved in ultrapure water (5 mg/mL) and eluted with ultrapure water at a fow rate of 0.5 mL/min.After freeze-drying, the powder obtain was DPSP-3 fraction.Te total polysaccharides content in each tube was determined by the phenol-sulfuric acid method.

Conformational Features Determination.
Fourier transform infrared (FT-IR) spectra analysis: the infrared spectrum was determined using the Nicolet iN10 microscope infrared spectrometer (Termo Fisher Technology Co., Shanghai, China).100 mg of KBr was added to 1 mg of polysaccharides and then ground and pressed.Infrared fullwavelength mode spectral scanning was performed in the frequency range of 4000 to 400 cm −1 .
Triple-helix structure analysis: Congo red solution (2 mL, 80 μmol/L) and polysaccharides solution (2 mL, 2.5 mg/mL) were mixed at a ratio of 1 : 1. NaOH solution was added to make the fnal concentration of NaOH with 0.0-0.5 mol/L.Te maximum absorption wavelength was measured by using the UV-vis spectrophotometer (TU-1810PU, Bejing Purkinje General instrumental factory, Beijing, China).

Scanning Electron Microscopy (SEM) Observation.
Scanning electron microscopy (SEM) (Carl Zeiss AG, Oberkohen, Baden-Wurttemberg, Germany) was used to observe the polysaccharides microstructure.Te polysaccharides were evenly coated on the carrier platform and before being coated with a thin layer of gold using a sputter coater (MC1000, Hitachi Limited, Tokyo, Japan).Te goldsprayed sample was placed in a scanning electron microscope, and the morphology was observed at 500x 1000x 2000x magnifcation.Ten, cells were incubated in the DMEM culture medium with 200, 400, and 800 μg/mL PSP, DPSP-3 solution, or LPS (25 μg/mL).After 24 h incubation, all culture medium was carefully removed, followed by the addition of 100 μL DCFH-DA (10 μM) dissolved in the serum-free medium for 30 min at 37 °C.Ten, the supernatant was removed, and the cells were washed three times with PBS.Fluorescence intensity was immediately detected at 485 nm excitation and at 525 nm emission using a microplate reader (Multiskan FC, Termo Fisher Scientifc, USA).Te RAW264.7 cells (1 × 10 5 cells/mL) were pipetted into 24-well plates, which were then incubated with 200, 400, and 800 μg/mL PSP, DPSP-3 solutions, or LPS (25 μg/mL).Te samples were centrifuged for separation (2,000 rpm, 10 min), and the supernatant was collected for the subsequent experiments.Te release of NO was determined by using the NO determination kit (Jiancheng bioengineering institute, Nanjing, China) according to manufacturer's instruction.Te release of TNF-α and IL-6 was determined by using commercial ELISA kits (Mlbio, Shanghai, China) according to the manufacturer's instructions.

Statistical Analysis.
All experimental data were expressed as means ± standard deviation (SD).Statistical calculations and graphing were performed using GraphPad Prism 8.0.2.SPSS 20.0 was used to evaluate statistical signifcance, and p < 0.05 was considered statistically signifcant.

. Results and Discussion
3.1.Isolation and Purifcation.Te yields of PSP and DPSP were (10.21 ± 0.50)% and (15.62 ± 0.71)% (w/w), respectively, and the yield of DPSP was 1.53 times that of PSP.Wang found that the optimal extract rates of favonoids from Moringa oleifera leaves by DES were 1.17 times and 1.42 times that of ethanol extraction and water extraction, respectively, and favonoids by DES improved antioxidant, antibacterial, and antitumor activities [24].Wu extracted acidic polysaccharides from lotus leaves with DES, and the results showed that the maximum yield (5.38%) was 1.67 times that of polysaccharides prepared with the pharmacopoeia method [25].Tese results were consistent with this study.
DPSP solution was eluted by DEAE-Sepharose Fast Flow column, and three peaks were obtained when concentration of NaCl increased to 0.4 mol/L in the elation, as illustrated in Figure 2  Journal of Food Quality

Molecular Weight Analysis.
Molecular weight afected the absorption of polysaccharides in vivo by afecting the physicochemical properties of polysaccharides such as solubility and viscosity [26].Some studies showed the high molecular weight usually inhibits its absorption, yet low molecular weight compounds were unable to form active triple-helix conformation [27].As shown in Figures 3(a) and 3(b), PSP and DPSP contained more than one fraction due to the presence of multiple peaks in the chromatogram.Te molecular weight of PSP ranged from 9.4 × 10 7 to 6.9 × 10 3 Da, and DPSP had four main peaks which were 4.1 × 10 7 , 7.7 × 10 5 , 7.9 × 10 3 , and 1.4 × 10 3 Da, respectively.Molecular weight analysis of PSP, DPSP, and DPSP-3 showed that the DES extraction method could degrade polysaccharides.As shown in Figure 3(c), DPSP-3 showed a single symmetric peak (3.2 × 10 6 Da) with a higher molecular weight.Wang found that the immunological activity of P. sibiricum polysaccharides was related to the molecular weight and main composition of monosaccharide, the immunological activity of high molecular weight polysaccharides was higher than that of low molecular weight polysaccharides, and the higher ratio of Gal and Rha signifcantly improved the phagocytosis in RAW264.7 cells [20].

Monosaccharide Composition Analysis.
Diferent monosaccharide compositions have an important infuence on the immunomodulatory activity of polysaccharides.Te study showed that the more complex the monosaccharide composition was, the better the biological activity of polysaccharides displayed [28].Helicteres angustifolia L. polysaccharides with high ratios of arabinose, galactose, xylose, and uronic acid signifcantly enhanced the proliferation of macrophages and stimulated the macrophages phagocytic capacity, as well as induced NO and immunomodulatory cytokines [29].Passifora foetida polysaccharides with high content of mannose (48.83%) could promote the release of NO, TNF-α, and IL-6 from macrophages [30], and the high content of mannose could contribute to the mild immunostimulatory activity [28].Te monosaccharide composition of PSP, DPSP, and DPSP-3 is shown in Figure 4. Tese polysaccharides consisted of mannose, rhamnose, glucose, galactose, arabinose, and a small number of glucuronic acid and galacturonic acid.
Results showed that DPSP and DPSP-3 had more mannose, rhamnose, galactose, and galacturonic acid, yet less glucose and glucuronic acid.After purifcation, the galactose content of DPSP-3 was 65.75%, which was 13.45 times that of the PSP.Te mannose content of DPSP-3 was 19.76%, which was 1.65 times that of the PSP.Te rhamnose content of DPSP-3 was 4.48%, which was 16 times that of the PSP.corresponds to C-H stretching vibration absorption [32], while peak at 1371 cm −1 corresponds to C-H bending vibration absorption.Tese three absorption peaks are characteristic peaks of polysaccharides.Te absorption peak at 1631 cm −1 corresponds to C=O bending vibration [33].

Conformational Analysis.
Te absorption peak at 1015 cm −1 corresponds to characteristic peaks of C-O-C of glucopyranose, which indicates that the three polysaccharides all contain pyranoside bonds [34].Meanwhile, absorption peak at about 889 cm −1 corresponds to β-glucosidic bonds [35], and DPSP-3 was higher than the other two polysaccharides, indicateding that it contained more β-glucoside bonds.Some studies showed that polysaccharides with β confguration had higher activity [26].As seen from Figure 5(b), after the maximum absorption, wavelengths showed obvious red-shift phenomenon, while with the increase of NaOH concentration, λmax did not decrease signifcantly.Tese results indicated that three polysaccharides had typical triple-helix conformation and were relatively stable at the concentration of 0-0.4 mol/L NaOH.Studies showed that polysaccharides changed to single-fexible chains, resulting in decreasing bioactivity [36].Previous research showed that the triple-helix structure of P. sibiricum polysaccharides was stable at the concentration of 0-0.4 mol/L NaOH [37], suggesting that DPSP might have higher and more stable biological activity.

SEM Analysis.
Te SEM images of PSP, DPSP, and DPSP-3 were shown in Figure 6.PSP presented a fat, faky, and rough surface.Although DPSP also had a fake and rough morphology, it also had a lot of independent spherical convex.DPSP-3 presented a curly shape and had smooth, porous, and lamellar surface with a few spherical edges.Research showed that acid sugar with large molecules had negatively charged, which resulted in increasing repulsive force between the molecules [38].Te smooth surface of polysaccharides probably had negative efect on the rehydration performance to reduce its solubility, which accounted for the lower solubility of some polysaccharides [39].Terefore, compared with the fat and faky structure, the curly structure of DPSP-3 predicted better biological activity.It indicated that diferent extraction and purifcation methods changed the morphological characteristics of polysaccharides.

Te Efects on the Cell Viability of RAW264.7 Cells.
Figure 7(a) showed the efects of PSP and DPSP-3 on the cell viability of RAW264.7.Tey showed no signifcant toxicity to RAW 264.7 cells at 100-1000 μg/mL.By contrast, in the concentration range of 200-1000 μg/mL, although two polysaccharides promoted cellular proliferation, concentration of 1000 μg/mL showed the lower proliferation efect.Terefore, polysaccharides concentration at 200, 400, and 800 μg/mL was chosen for the subsequent immunomodulatory activity experiments.Cells.Phagocytosis of macrophages is the most basic regulatory mechanism in the immune response and one of the main characterizations of its immunity activation.Phagocytosis can be divided into three steps: recognition of foreign substances, formation of phagosomes, and maturation of phagosomes [40].Te phagocytosis of macrophages can refect the activation degree of macrophages, and the phagocytosis of neutral red can simply and quickly refect the phagocytosis of macrophages.As shown in Figure 7(b), the phagocytosis ability of RAW264.7 cells signifcantly enhanced dose-dependently.DPSP-3 was signifcantly higher than PSP in three concentrations.At high concentration (800 μg/mL), the PSP group of phagotrophic rate was 1.78 times that of the control group, the DPSP-3 group of phagotrophic rate was 3.09 times that of the control group.Tis might be attributed to the fact that the immunomodulatory activity of the polysaccharides was related to the molecular weight and the major monosaccharide composition.Meanwhile, the higher Gal and Rha content in DPSP-3 had an important efect on the improvement of the phagocytosis capacity of RAW264.7 cells [28].Macrophages are one of the most important immune cells, and the activated macrophages can eliminate pathogens or tumor cells by phagocytosis or secretion of proinfammatory factors [21,44].As can be seen from Figures 8(c) and 8(d), PSP and DPSP-3 signifcantly stimulated the release of IL-6 and TNF-α in RAW264.7 cells at 200-800 μg/mL.Te release of IL-6 was measured as (567.28 ± 20.44) and (602.16 ± 16.66) pg/mL at 200 μg/mL, respectively, which were 19.67 times and 20.88 times that of the control group.TNF-α showed a signifcant dosedependent relationship with PSP and DPSP-3.At 800 μg/ mL, the release of TNF-α was (165.71 ± 6.01) and (227.16 ± 5.29) pg/mL, respectively, which were 3.80 times and 6.35 times that of the control group.Compared with PSP, DPSP-3 could signifcantly improve the release of TNFα by about 37%.

Conclusion
In this study, we isolated and purifed polysaccharides from P. sibiricum and analyzed their structural characterization and immunomodulatory activity in RAW264.7 cells.Te yield of DPSP was (15.62 ± 0.71)%, and its yield was 1.53 times that of PSP.DPSP-3 purifed from DPSP at 0.04 M NaCl showed molecular weight of 3.2 × 10 6 Da, and the content of galactose, mannose, and rhamnose was 13.45, 1.65, and 16.00 times that of PSP, respectively.Both PSP and DPSP-3 signifcantly improved phagotrophic capacity and increased the release of ROS, NO, IL-6, and TNF-α in RAW264.7 cells.Meanwhile, DPSP-3 exhibited better immunomodulatory activity, which might be related to the higher content of mannans, galactose, β-glucosidic, bonds, and triple-helix structure in DPSP-3.Based on the speculation of the relationship between the structure and immunomodulatory activity of P. sibiricum polysaccharides in this study, the immune-related receptors and pathways can be studied in the future.In general, DES can not only improve the yield of polysaccharides but also change the structure and activity of polysaccharides.However, the principle of changing polysaccharides structure and the mechanism of enhancing polysaccharides activity are not clear.Tese questions need to be further explored.Tis study developed an efcient method for preparing polysaccharides from P. sibiricum utilizing DES.Te results showed that DES was an efcient solvent for polysaccharides extraction, and DPSP-3 could be used as potential immunomodulatory agents in the felds of health food industry.
mL) were loaded into 96-well plates.After 24 h incubation, DEME culture medium, LPS (25 μg/mL), or diferent concentrations of 200, 400, and 800 μg/mL, PSP and DPSP-3 solutions were pipetted into each well for another 24 h incubation.Te medium was carefully removed, and 100 μL of 0.1% neutral red was added into each well and incubated for 1 h.After washing three times with PBS, each of the wells was loaded with 100