Chemical Structure and Immune Activation of a Glucan From Rhizoma Acori Tatarinowii

Rhizoma Acori Tatarinowii is a traditional Chinese herb used to treat depression and coronary heart disease. Studies on its active components mainly focus on small molecular compounds such as asarone and other essential oil components, while the large molecular active components such as polysaccharides are ignored. In this study, we aimed to study the chemical structure and immune activation of polysaccharides from Rhizoma Acori Tatarinowii. In this study, a polysaccharide (RATAPW) was isolated and purified by DEAE-52 cellulose and Sephadex G-100 column chromatography from alkali extraction polysaccharide of Rhizoma Acori Tatarinowii. The average molecular weight of RATAPW was 2.51 × 104 Da, and the total carbohydrate contents of RATAPW were 98.23 ± 0.29%. The monosaccharide composition, methylation, and nuclear magnetic resonance (NMR) analysis results displayed that the polysaccharide was α-1,4-glucan with short α-1,6 branches. Immunofluorescence assay and inhibitor neutralization assay indicated that RATAPW could promote the TNF-α production of RAW264.7 macrophage through the nuclear factor kappa B (NF-κB) molecular signaling pathway. Treatment with 200 μg/ml of RATAPW enhanced a 38.77% rise in the proliferation rate of spleen lymphocytes. RATAPW also enhances ConA-induced T cells and lipopolysaccharide (LPS)-induced B cell proliferation in a dose-dependent effect. Our study lays a foundation for the discovery of natural polysaccharide immune modulators or functional food from Rhizoma Acori Tatarinowii.


INTRODUCTION
Herb medicine Rhizoma Acori Tatarinowii has appetizing, sedative, detumescent, and analgesic effects and is often used to treat joint pain, depression, and Alzheimer's disease syndrome (1). To date, Rhizoma Acori Tatarinowii has been studied for various biological activities, such as potentiating neuronal differentiation of PC12 cells (2), inhibiting the proliferation of cancer cells (3), and promoting the absorption and transport of compounds by inhibiting glycoprotein (4).
However, studies on the active components of Rhizoma Acori Tatarinowii mainly focused on the small molecular compounds such as asarone and other essential oil components but ignored the large molecular components such as polysaccharides. In recent years, natural polysaccharides attracted extensive attention for their immunomodulatory activities (5). For example, a polysaccharide from the fruiting bodies of Helvella leucopus induced macrophage activation via the nuclear factor kappa B (NF-κB) signal pathway (6). The α-glucan from ginger could activate RAW264.7 cells to release cytokines (7).
Inspired by these studies, we speculated that the immune activity of Rhizoma Acori Tatarinowii may be related to the active polysaccharide components. Consistently, our previous studies showed that the polysaccharides obtained by water extraction have prominent immunological activity (8). However, the extraction yields, structures, and bioactivities of polysaccharides are strictly affected by the extraction process (9). Studies have shown that polysaccharides obtained by different extraction methods vary greatly. He et al. showed alkalic-extractable polysaccharides from Ziziphus jujuba cv. with higher antioxidant activity (10). In this study, we aimed to study the chemical structure and immune activation of polysaccharides from Rhizoma Acori Tatarinowii. We extracted the polysaccharide from the Rhizoma Acori Tatarinowii with the alkaline solution. The crude polysaccharide was isolated and purified by anion exchange and gel filtration chromatography. The chemical structure was characterized by monosaccharide compositions, infrared spectra (IR), methylation, and nuclear magnetic resonance (NMR) analyses. The immune activation of the polysaccharide was determined by enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and inhibitor neutralization assay.

Isolation and Purification of Polysaccharide From Rhizoma Acori Tatarinowii
The dried Rhizoma Acori Tatarinowii was grounded into powder and then defatted with 95% ethanol. The solid residues were extracted with distilled water (1:10, w/v) at 80 • C for 2 h, and this extraction process was repeated 3 times. The supernatants were pooled, collected, and studied previously (8). To obtain more kinds of polysaccharides, we further extracted polysaccharides from the residue by alkali extraction and alcohol precipitation. The solid residue was extracted 3 times with 0.2 M sodium hydroxide at 50 • C for 2 h. Subsequently, the collected supernatants were concentrated, neutralized, and precipitated with 4 times absolute ethyl alcohol. The polysaccharide precipitates were collected and redissolved in distilled water. Then, the polysaccharide solution was dialyzed for 72 h in distilled water (change the water every 4 h and Mw cutoff: 3,500 Da). After freeze-drying, the alkali extracted crude polysaccharide was obtained. The crude polysaccharide was dissolved in deionized water, and the mixture was centrifuged. The supernatant was loaded on DEAE-52 cellulose (5 cm × 50 cm, Cl − form) and eluted with distilled water and different concentrations of gradient NaCl solution (0.1, 0.2, and 0.5 M NaCl) at a constant flow rate consecutively. The total carbohydrate content of each fraction was detected using the phenol-sulfuric acid method (11). According to the solubility and content of polysaccharides, water elute was further purified using a Sephadex G-100 column. The collected components were eluted with distilled water at a flow rate of 0.2 ml/min. The eluent in the test tube with absorbance greater than 0.3 was collected, lyophilized, and named RATAPW.

Infrared Spectra Analysis
An amount of 2 mg dry RATAPW was mixed with 50 mg chromatographic pure KBr. Agate pestle and mortar were used for grounding the sample, and it was further analyzed using the Fourier transform infrared spectrophotometer (BRUCK, Germany) after pressing in pellets (13). The measurement wavenumber region ranged from 400 cm −1 to 4,000 cm −1 .

Chemical Component and Monosaccharide Composition Analysis
The phenol-sulfuric acid method and Bradford's method (14) were used to determine the total sugar and protein contents, respectively.
An amount of 10 mg RATAPW was hydrolyzed using the 3 M TFA at 120 • C for 3 h (15). The hydrolysate was washed three times with methanol and evaporated. Finally, the hydrolyzed material was dissolved with 5 ml of deionized water, transferred to a 50 ml volumetric flask, and diluted to 50 ml. High-performance anion-exchange chromatography (HPAEC) equipped with ICS-5000 (Waltham, MA, United States) and a CarboPac TM PA-20 analytical column (3 mm × 150 mm) was employed. Here, 15 mM NaOH and 100 mM sodium acetate were used as mobile phases for gradient elution at 0.3 ml/min (16). The monosaccharide kinds of RATAPW were determined by comparing the retention times with fifteen monosaccharide standards (fucose, galactosamine, rhamnose, arabinose, glucosamine, galactose, glucose, N-acetyl-D-glucosamine, xylose, mannose, ribose, galacturonic acid, guluronic acid, glucuronic acid, and mannuronic acid).

Methylation and Glycosidic Linkage Analysis
The methylation analysis method was applied to confirm the glycosidic linkages of RATAPW based on the reference (17). In brief, the dried polysaccharide RATAPW (3 mg) was dissolved in 1 ml dried DMSO and dried NaOH powder, ultrasonically treated for 1 h. Subsequently, the methyl iodide was added under dark conditions and stirred at 30 • C for 1 h to obtain the methylated product. Further hydrolyzed with 4 M TFA, reduced with NaBH 4 , and acetylated with acetic anhydride. Ultimately, the partially methylated alditol acetates (PMAA) containing the linkage type of RATAPW were analyzed by chromatography-mass spectrometry (GC-MS) system (Shimadzu GCMS-QP 2010) equipped with an RXI-5 SIL MS column (30 m × 0.25 mm × 0.25 µ m).

Nuclear Magnetic Resonance Analysis
A Bruker 600 MHz spectrometer equipped with a 1 H/ 13 C double probe was used for NMR analysis at 25 • C (18). An amount of 50 mg dried RATAPW was dissolved in 1 ml D 2 O and frozen three times to exchange H protons into deuterium completely. The lyophilized sample was then dissolved in 0.5 ml D 2 O overnight before NMR analysis. 1 H NMR, 13 C NMR, 1 H-1 H COSY, HSQC, HMBC, and NOESY spectra of RATAPW were recorded.

Measurement of TNF-α Cytokine Production Induced by RATAPW
The RAW264.7 cell was a kind gift from Prof. Jinyou Duan at Northwest A&F University. In 96-well plates, RAW 264.7 cells (1 × 10 5 cells/well) were incubated with different concentrations (50, 100, or 200 µg/ml) of RATAPW for 24 h. phosphate buffer saline (PBS) was used as the negative control and lipopolysaccharide (LPS) (2 µg/ml) was used as the positive control. The cell supernatants were collected. TNF-α proteins in the cell supernatants were measured using the ELISA kits (R&D) according to the instructions (19). To determine the effect of RATAPW on the growth profile of RAW264.7 cells, 0.5 mg/ml MTT was added to the plates and further incubated for 4 h at 37 • C. The optical density was measured at 570 nm.
In addition, RAW 264.7 cells were pretreated with PBS or 3 µM BAY 11-7082 for 1 h and incubated with 200 µg/ml RATAPW or LPS (2 µg/ml) for 24 h. TNF-α cytokine content in the different wells was measured using the ELISA kits.

Immunofluorescence Tests for NF-κB Activation
After being treated with 200 µg/ml RATAPW or LPS (2 µg/ml) for 3 h, the primary NF-κB p65 antibody was added to RAW 264.7 cells for 1 h and then incubated with a FITC-labeled second antibody for 1 h. Finally, we added DAPI and viewed green p65 protein and blue nuclei fluoresce by laser confocal microscopy.

Effect of RATAPW on Lymphocyte Proliferation
The spleen lymphocyte suspension was obtained by grinding, sieving, and lysis of erythrocytes. Notably, 1 × 10 7 cells/well lymphocyte cells in 96-well plates were incubated with different concentrations of RATAPW or pre-added the mitotic inducer Con A (5 µg/ml) and LPS (2 µg/ml). After 48 h, the MTT method was used to evaluate the effect of RATAPW on lymphocyte proliferation in vitro.

Statistical Analysis
Dates were expressed as mean value ± standard deviation. Oneway and two-way ANOVA were used for statistical significance analysis using the GraphPad Prism 8.0 software.

Alkali Extraction and Column Chromatography Purification of Polysaccharide
The crude polysaccharide was isolated from dried Rhizoma Acori Tatarinowii through alkali extraction (Figure 1). A polysaccharide designated RATAPW was obtained after DEAE-52 cellulose ion-exchange chromatography and gel filtration chromatography purification steps. The yield of RATAPW was 2.41 ± 0.27% from the crude polysaccharide.

Molecular Weight and Infrared Spectral Analysis
The HPGPC peak revealed that the polysaccharide RATAPW was homogeneous and of high purity, with only one symmetrical absorption peak (20). The weight-average molecular weight (Mw) of RATAPW was 2.51 × 10 4 Da (T: 41.803 min) according to the calibration curve (Figure 2A). In Figure 2B

Chemical and Monosaccharide Composition of RATAPW
The total sugar contents in this fraction were 98.23 ± 0.29% after lyophilization. Only a trace amount of protein (1.52 ± 0.07%) was measurable in RATAPW. HPAEC results showed that RATAPW was mainly composed of glucose (Figure 3).

RATAPW Induced RAW 264.7 to Produce TNF-α via the NF-κB Pathway
The MTT results showed that different concentrations of RATAPW did not promote the proliferation of macrophages (Supplementary Figure 1). As shown in Figure 6A, TNF-α production of RAW 264.7 cells was significantly increased by RATAPW with a dose-dependent effect. Endotoxin contamination results showed that the endotoxin content is less than 0.01 EU, which ensures that the effect of RATAPW on RAW 264.7 cells was not due to endotoxin contamination (23).   Nuclear factor kappa B is a pivotal nuclear transcription factor, which is associated with multiple immune genes and manipulated the cytokine responses (24). External irritants can cause the inactive IκBα in the cytosolic phosphorylation and degradation and then induce p65 subunit translocation to the nucleus (25,26). Thus, we detected the NF-κB activation in RAW264.7 cells by immunofluorescence assay. Results showed that RATAPW and LPS could activate NF-κB and make cytoplasmic NF-κB p65 protein translocated to the nucleus ( Figure 6B). Furthermore, to validate whether the NF-κB activation was involved in RATAPW-induced cytokine production, 3 µM BAY 11-7082 (an NF-κB inhibitor) was added before adding RATAPW or LPS. Experimental results showed that BAY 11-7082 overtly suppressed TNF-α secretion in RAW264.7 cells (Figure 6C). All these results show that RATAPW can activate macrophages via the NF-κ B pathway.

RATAPW Promotes Splenocyte Proliferation
As a major site of immune responses, the induction and regulation of spleen proliferation are very important for the immune system (27). Mouse primary splenocytes are mainly composed of T and B cells (28). Our results showed that RATAPW promoted the proliferation of spleen lymphocytes (Figure 7A). It also acted in concert to synergistically enhance ConA-stimulated T cells and LPS-induced B cell proliferation in a dose-dependent effect (Figures 7B,C).

DISCUSSION
In this study, a polysaccharide named RATAPW (molecular weight: 2.51 × 10 4 Da) was isolated from Rhizoma Acori Tatarinowii with alkali extraction and alcohol precipitation, and its primary chemical structure, physicochemical properties, and immune activity were characterized. The HPAEC results showed that RATAPW was mainly composed of glucose, combined with methylation analysis results and NMR spectra, and RATAPW was an α-1,4-glucans branched at C-6 every 32 glucose residues. The bioactivity tests showed that RATAPW could activate  the RAW 264.7 cells increasing TNF-α secretion by NF-κB pathways. Besides, RATAPW can also enhance T and B lymphocyte proliferation.
Glucans are divided into αand β-conformation, and the linkage of the glucose can be either 1,4 or 1,6 glycoside bonds (29). Reports suggested that the chemical structure and chain conformation of glucan directly affect its biological activity (30). Therefore, it is necessary to detail the structure-activity relationships between glucans and immunoregulation. Till now, it has been known that many natural β-glucans exhibit significant immunomodulatory activities (31). A set of receptors for βglucans have been revealed, such as toll-like receptors (TLRs), dectin-1, complement receptor 3 (CR3), and lactosylceramide (30). Similarly, a lot of studies have demonstrated that α-glucans also had kinds of immune activities, which can be directly influenced by the solubility, molecular weight, molecular charge, branching degree, and glycosidic bonds of α-glucans (32).
Reports suggest that the (1→3)-, (1→4)-, or (1→6)-αglucans can stimulate immune cells to secrete cytokines to different degrees (33). Consistently, our results showed that RATAPW was an α-1,4-glucan with few (1→6) branches and can activate macrophages to release cytokines. Our previous study has displayed that two α-glucans from Radix Paeoniae Alba with a similar structure to RATAPW can also promote splenocyte proliferation and RAW264.7 phagocytic activity (34). The molecular weight is another important impact factor for macrophage stimulation and the production of cytokines. The molecular weight of RATAPW was 25.1 kDa, within the range of 10 kDa to 1,000 kDa. Previous reports have indicated that polysaccharides with molecular weights in this range have the highest immunoregulatory activities (35). The solubility can also affect the recognition of polysaccharides by the antigenpresenting cells (APCs) and cytokine production (36). It has been demonstrated that the increase of water solubility of α-glucans enhances its immunostimulant activity (37). The immune activity of RATAPW might also be linked to its high solubility. Therefore, it was speculated that the immunoregulatory activity of RATAPW might be attributed to its high solubility, moderate molecular weight, and low degree of branching.
Glucans are toll-like receptor 4 (TLR4) ligands on the macrophages' cell surface and activate immune cells through the TLR4/IKK/NK-κB pathway (38). α-Glucan could also activate the MAPK signaling pathway to promote the secretion of cytokines (9). Our results proved that RATAPW activates the RAW 264.7 cells and increases TNF-α secretion by the NF-κB pathways. The possible receptor and molecular pathway of RATAPW need to be further explored. In general, the glucan RATAPW could be explored as an immunomodulator in the field of health food and medicine.

DATA AVAILABILITY STATEMENT
The original contributions presented in this study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
WZ designed the research, performed the experiments, analyzed the data, and wrote the manuscript. JH and YH performed the experiments. JL analyzed the experimental data. JZ analyzed the NMR data. PL supervised the work and wrote and reviewed the manuscript. All authors contributed to the article and approved the submitted version.