Structural elucidation of a novel arabinogalactan LFP-80-W1 from Lycii fructus with potential immunostimulatory activity

Polysaccharides are the most important effective components of Lycii fructus, which has a variety of biological activities and broad application prospects in the fields of medicine and food. In this study, we reported a novel arabinogalactan LFP-80-W1 with potential immunostimulatory activity. LFP-80-W1 was a continuous symmetrical single-peak with an average molecular weight of 4.58 × 104 Da and was mainly composed of arabinose and galactose. Oligosaccharide sequencing analyses and NMR data showed that the LFP-80-W1 domain consists of a repeated 1,6-linked β-Galp main chain with branches arabinoglycan and arabinogalactan at position C-3. Importantly, we found that LFP-80-W1 could activate the MAPK pathway and promote the release of NO, IL-6, and TNF-α cytokines in vitro. Therefore, our findings suggest that the homogeneous arabinogalactan from Lycii fructus, can be used as a natural immunomodulator.

Lycii fructus, a famous traditional Chinese medicine tonic for thousands of years, belongs to the dried ripe fruit of Lycium barbarum L (5). As a functional food and drug, it is widely used worldwide as a popular nutritional food or healthy dietary supplement, appearing in various forms in fruit juice, wine, tea and various solid foods (6). Lycii fructus polysaccharides (LFPs) have been proven to be the most famous bioactive ingredients of Lycii fructus, which possess antiaging, antidiabetic, antifibrotic, neuroprotective, and immunomodulation properties (7). At the same time, the immunomodulatory effect is primary and is involved in other activities (8). The chemical structure and composition of LFPs have great diversity, and subtle structural changes may affect their immune activity. As such, the immune activity of LFPs is influenced by their monosaccharide composition, molecular weight, and branching degree (9). LBPF4, a polysaccharide-protein complex mainly composed of arabinose and galactose (1.50:2.50), was found to enhance macrophage function by enhancing phagocytosis and activating the NFκB pathway (10). Likewise, a previous study reported that higher molecular weight polysaccharides exhibited better immunomodulatory activity than lower molecular weight polysaccharides extracted from Lycii fructus (11). Additionally, a study has shown that the immunocompetent domains of arabinogalactan LRGP3 seem to be related to the branching degree of the side chain. Compared with the galactan skeleton, the arabinan side chains had stronger macrophage activation activity. In contrast, the inner galactan core significantly increased the complement-fixating activity (12). However, which monosaccharide species or connective structures are closely related to the immune activity is unclear. Therefore, the relationship between the clear structure and immunomodulatory activities of LFPs needs to be further investigated.
In our ongoing search for bioactive polysaccharides, a uniform novel polysaccharide LFP-80-W1 was isolated and purified by column chromatography from Lycii fructus. Different chemical, spectroscopic, and oligosaccharide sequencing characterized its structure. In addition, the immunomodulatory effect of LFP-80-W1 on RAW 264.7 cells and its related target mechanism was also investigated. The results of this study provide useful information for illustrating the structure-function relationship of LFPs and developing immune-enhancing food and drugs from Lycii fructus.

Extraction, separation, and purification of LFP-80-W1
Air-dried Lycii fructus (10 kg) was defatted with 80% ethanol twice and then extracted twice with reflux water at 100 • C. The liquid supernatant was depressurized and concentrated, and the ethanol concentration was adjusted to 30% to precipitate insoluble components such as fiber and pectin. Subsequently, anhydrous ethanol was added to the supernatant to a final concentration of 80% and allowed to precipitate overnight. LFP-80 (163 g) was obtained by freezedrying and deproteinized by Sevage reagent. Crude LFP-80 (2 g) was redissolved in water and separated on a DEAE-52 cellulose column (4.5 cm × 60 cm) by anion-exchange chromatography. The water-washed fraction was collected and further purified by gel permeation chromatography Sephacryl S-100 column (150 cm × 2.6 cm). The earlier elution part was monitored and collected using the phenol-sulfuric method to obtain the target polysaccharide LFP-80-W1. The protruding peak of the elution curve is shown in Supplementary Figure 1.

Physicochemical property of polysaccharides
The protein content was determined using the Bradford method with bovine serum albumin as the reference (13). The Frontiers in Nutrition 02 frontiersin.org total sugar content was determined by the phenol sulfuric acid method with glucose as the reference (14).

Morphological analysis
According to previous literature reports (15,16), the morphological features of LFP-80-W1 were recorded using a scanning electron microscope (SEM) (Gemini 300, ZEISS, Germany). The acceleration voltage was set at 5.0 kV. LFP-80-W1 solution was dripped on the freshly cleaved mica surface, and scanning probe microscopy images were collected by Atomic force microscopy (AFM) (Dimension Edge, Bruker, Germany).

Homogeneity and molecular weight (MW) assays
The molecular weight assay of LFP-80-W1 was determined by high-performance gel permeation chromatography (HPGPC), which was performed on a Waters 2,695 HPLC system (Shimadzu, Kyoto, USA) equipped with a refractive index detector (RID) and two columns (TSK-gel G-3000 and 5000 PW, 8.0 × 300 mm, 6 µM, Showa Denko Co., Tokyo, Japan) connected in series at 35 • C. The sample was eluted with 0.01 M PBS buffer at a flow rate of 0.4 mL/min. Furthermore, the Mw was estimated with reference to the calibration curve prepared by standards.

Monosaccharide composition assays
The monosaccharides were simultaneously identified and quantitated according to a previously described method (17,18). Briefly, 5 mg LFP-80-W1 was liberated with 2 M TFA at 110 • C for 2 h. The released mixture of monosaccharides was then transformed to corresponding alditol acetate derivatives and separated by a Perkin Elmer Clarus 680 GC system (Perkin Elmer, CA, USA) equipped with an Agilent HP5-MS capillary column (30 m × 0.25 mm × 0.25 µm). The oven temperature was programmed from an initial 100 • C for 3 min to 200 • C at 20 • C/min and held for 2 min, then to 230 • C at 5 • C/min and held for 2 min, 280 • C at 10 • C/min for 8 min with helium as the carrier gas.

Glycosyl linkage composition analysis
To analyze the composition of glycosyl linkage of LFP-80-W1, methylation analysis was carried out. Briefly, 5 mg vacuumdried sample of LFP-80-W1 was ultrasonically dissolved in 4 mL DMSO and treated with 500 mg NaOH 0.5 mL of methyl iodide was added and stirred for 2.5 h in the dark. The reaction was inhibited by adding 2 mL of ultrapure water. The methylated polysaccharide was extracted into chloroform and lyophilized to obtain the methylated polysaccharide. The fully methylated products were converted into corresponding partially methylated alditol acetates (PMAAs) derivatives according to Needs' method (19). The PMAAs were then hydrolyzed with 2 M TFA at 110 • C for 2 h, reduced with NaBH 4 , and acetylated with pyridine and acetic anhydride. The acetylated PMAAs were analyzed and identified by the relative retention times and diagnostic GC-MS fragmentation patterns compared with CCRC standard spectral database and previously literature (20,21).

Partial hydrolysis and oligosaccharide sequencing analysis
In this study, ABEE-labeled oligosaccharide fragments were obtained by partial acid hydrolysis and derivatization, and their oligosaccharide sequences were analyzed by UHPLC-QTOF/MS. Briefly, LFP-80-W1 (4 mg) partially was hydrolyzed with 2 mL of 1 mol/L TFA at 70 • C for 2 h and the excess acid was removed after hydrolysis. The hydrolyzed sample was mixed with derivatization reagent (0.6 mol/L ABEE, glacial acetic acid and 1.4 mol/L ABEE). The detailed derivatization method was described above (22,23).

Immunomodulatory activity of LFP-80-W1
Cell culture RAW 264.7 cells were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained as mycoplasma-free sub-confluent monolayer cultures in RPMI-1640 with 20% FBS at 37 • C in a humidified 5% CO 2 incubator.
Frontiers in Nutrition 03 frontiersin.org Cell viability assay RAW 264.7 cells were seeded in 96-well plates at a density of 1 × 10 5 cells/mL in triplicate and incubated for 24 h. the cells were then exposed to a series of concentrations of LFP-80-W1 (0, 25, 50, 100, 200, and 400 µg/mL) in serum-free RPMI-1640 for 24 h. cell viability was detected by CCK-8 assay, which shows the mitochondrial activity of living cells by using a microplate spectrophotometer (bio Tek, USA). The absorbance of each well was measured at 450 nm. Cells only suffered medium change were used as vehicle control and normalized to 100% viability.

Measurement of NO, IL-6, and TNF-α cytokine production
The RAW 264.7 cells in the logarithmic growth phase were inoculated into a 12-well plate at 2.0 × 10 5 cells/mL for 24 h. The cells were then treated with lipopolysaccharide (LPS; 1 µg/mL) or LFP-80-W1 at different concentrations (10, 30, and 100 µg/mL) for 24 h or 48 h, respectively. The NO level in the culture supernatant was determined with a commercially available kit based on the Griess reaction. According to the manufacturer's instructions, the ELISA kit determined the cellular abundances of TNF-α and IL-6.

Real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis
Cells were plated in 6-well plates with 5 × 10 5 cells/mL. Total RNA was extracted using the RNeasy Mini Kit (Tiangen, Beijing, China) in an RNase-free environment. Approximately 1 µg RNA was reversetranscribed into cDNA using PrimeScript TM RT Master Mix (TaKaRa, Beijing, China). RT-qPCR was performed with the SYBR R qPCR Master Mix (Vazyme, Nanjing, China) (24). Ct values were determined after normalization to housekeeping gene GAPDH mRNA as the invariant control. All the reactions were repeated in three iterations, and relative gene expression multiple was calculated by the 2 − CT method. The primers (Sangon Biotech, Shanghai, China) used were indicated in Supplementary Table 1.

Western blot analysis
Cells were plated in 6-well plates with 5 × 10 5 cells/mL. The protein samples of the cells were lysed with RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. The samples were separated with 20 µg total protein and transferred to cellulose nitrate (NC) membrane at 4 • C. The membranes were blocked with TBST containing 5% skim milk for 1 h and probed with primary antibody (1:1,000) against ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK overnight at 4 • C, then incubated for an additional 1 h with the secondary antibody (1:5,000). The antibody signal was detected by Bio-Rad chemiluminescence imaging system (California, USA). Software ImageJ was used to quantify and normalize strip intensity.

Statistical analysis
All data are presented as mean ± SD of a minimum of three independent experiments performed in three biological replicates and analyzed by one-way ANOVA in SPSS 17.0 (SPSS Inc., Chicago, IL, USA) at similar conditions unless otherwise specified. The criterion for statistical significance were * p< 0.05, * * p< 0.01 and * * * p< 0.001.

Results and discussion
Purification, homogeneity, and composition of LFP-80-W1 Currently, two crude polysaccharides (LFP-30 and LFP-80) were isolated and extracted from the Lycii fructus by fractional alcohol precipitation with a yield of 1.32 and 1.67% based on dry matter. Water soluble polysaccharides LBP-80 were separated by DEAE-Sepharose column chromatography with water elution to obtain the neutral component. The refined LFP-80-W1 (with a recovery rate of 7.50%) was further isolated and purified using Sephacryl S-100. The total polysaccharide and protein content was estimated to be 94.50 and 1.70% by classical phenol-sulfuric acid assay and Bradford method, respectively. LFP-80-W1 showed a continuous symmetrical single peak on HPGPC, and its MW was calculated to be 4.58 × 10 4 Da by comparing it with the standard dextrans ( Figure 1A). LFP-80-W1 was mainly composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose with a molar ratio of 2.97:45.00:3.65:1.06:6.48:40.85, of which arabinose and galactose were the main monosaccharides ( Figure 1B). The monosaccharide composition was similar to that of polysaccharides isolated from Lycii fructus as previously reported (25,26), suggesting that arabinose and galactose may be common monosaccharides commonly contained in Lycii fructus. However, they have structural diversity likely due to different external environments and extraction protocols.

Methylation analysis of LFP-80-W1
Methylation of polysaccharides is one of the powerful methods to analyze and identify the molecular structure of polysaccharides, which can provide the type and proportion of glycoside bonds. The GC-MS spectra of the PMAAs of LFP-80-W1 are shown in Figure 1C. The results of methylation analysis demonstrated that LFP-80-W1 contained 11 partially methylated glycolol acetate peaks (Supplementary Figure 3 for mass spectra of the targeted peaks). As summarized in Table 1   residues. In addition, LFP-80-W1 contains numbers of linear polysaccharides, from which it can be inferred that its structure was composed of the main chain and several branches. Some methylation peaks were not accurately identified, probably because of undermethylation or introduced by precolumn derivatization. However, the overall glycosyl linkage composition was consistent with the monosaccharide composition.

Morphology analysis of LFP-80-W1
SEM was the most commonly used technique to investigate the microstructure at a submicron level. SEM obtained the Frontiers in Nutrition 05 frontiersin.org surface morphology of LFP-80-W1, and the results are shown in Figure 2A. LFP-80-W1 showed an irregular thin fragment ship with a loose structure, suggesting the presence of a complex polysaccharide branching structure. AFM analysis is a detection method to directly observe the conformation and three-dimensional surface states of biological macromolecules. The AFM images of LFP-80-W1 are shown in Figure 2B.
In the 5 nm field of view, LFP-80-W1 primarily comprised random linear chains and a few spherical aggregations, with conical shapes of varying heights in the 3D image, indicating that intramolecular and intermolecular van der Waals forces and hydrogen bonding resulted in the assembly of LFP-80-W1 molecules into aggregate structures.

NMR spectroscopy analysis of LFP-80-W1
NMR analysis further characterized the structure of LFP-80-W1. In the 1 H-NMR spectrum (Figure 3A), 11 anomeric signal peaks located at δ 4.35-5.27 were designated as glycosylated residues A-K, and it can be founded the corresponding 11 anomeric carbon protons 13 C-NMR (Figure 3B) spectrum.  NMR spectra recorded for LFP-80-W1 (600 MHz): (A) 1 H NMR spectrum; anomeric hydrogen signals are annotated. (B) 13 C NMR spectrum; anomeric carbon signals are annotated. (C) HSQC spectrum. The most relevant correlations are annotated and marked in gray. A1 represents the cross-peak between H-1 and C-1 of residues A; B1 represents the cross-peak between H-1 and C-1 of residues B and so on. (D) HMBC spectrum. The most relevant correlations are annotated and marked in gray. A1-D5 represents the cross-peak between H-1 of residues A and C-5 of residues D; J6-A1 represents the cross-peak between H-6 residues J and C-1 of residues A and so on. (Glycosylated residues A-K are summarized in Table 2).    The C/H chemical shifts of each glycosylated residue were classified in combination with 2D-NMR spectra and are listed in Table 2. The structural similarity of sugar units caused the signal overlapping in the resonance crowded region, and hindered the further structure resolution of LFP-80-W1. According to the existing information, we have assigned the parts without overlap. As the emergence of unambiguous approaches, more refined polysaccharide structures will be revealed in the future.

Structure confirmation by oligosaccharide profiles analysis
The dissociation of polysaccharides into constitutive and measurable oligosaccharides has been the critical component for characterizing polysaccharides (32). In order Frontiers in Nutrition 09 frontiersin.org  to further confirm the structure of LFP-80-W1, the sequencespecific oligosaccharide fragments obtained were used for polysaccharide identification by tandem mass spectrometry.
Compared with enzymatic hydrolysis, partial acid hydrolysis can determine the main chain structure of polysaccharide. Peng et al. obtained arabinan oligomers with 2-7 DP from LRGP3, and ESI-MS analysis showed that the high stability of arabin oligomers toward enzymatic hydrolysis (12). However, in this experiment, there was no arabin oligomers of side chain, only the terminal arabinose was found. Therefore, more information about polysaccharide backbone galactans can be obtained by partial acid hydrolysis.
Combining all above results, the probable structure detail of LFP-80-W1 was designed in Figure 5. The structure of the fraction is mainly a carbohydrate chain containing a highly branched arabinogalactan. Its structure was consistent with the backbone of 1,6-substituted linear galactan. Furthermore, the neutral arabinogalactan and arabinan organized the bushy sidechains at C-3 of Galp along the backbone axis.

Immunomodulatory activity of LFP-80-W1
Cell viability of LFP-80-W1 on RAW 264.7 cells As shown in Figure 6D, after the treatment with LFP-80-W1 for 24 h, the cell viability rate of RAW 264.7 cells in the concentration range of 25-400 µg/mL was significantly increased in a dose-independent relationship compared with that of the normal control group (P < 0.05). When LFP-80-W1 concentration reached 100 µg/mL, the cell viability effect was the most significant, and cell viability began to decline when concentrations began to exceed 100 µg/mL. Thus, polysaccharide concentrations less than 100 µg/mL were used for further investigation.

Measurement of NO, IL-6, and TNF-α cytokine production
The secretion of NO, IL-6, and TNF-α in the conditioned medium of RAW 264.7 cells was quantified by ELISA assays to evaluate whether LFP-80-W1 could activate RAW 264.7 cells. Firstly, LFP-80-W1 (10, 30, and 100 µg/mL) could promote NO production in a dose-dependent manner whether treated for 24 h or 48 h ( Figure 6A). As shown in Figure 6B, TNFα were significantly increased in cells produced by different concentrations of LFP-80-W1 and LPS compared with the control group (P < 0.05). IL-6 was enhanced when the cells were treated with 30 and 100 µg/mL LFP-80-W1 and LPS at different times ( Figure 6C). These results suggest that LFP-80-W1 can activate RAW 264.7 cells to secrete NO, IL-6, and TNF-α in vitro, and that the function of LFP-80-W1 was similar to that of LPS.

RT-qPCR analysis
To confirm the above observation, an RT-qPCR assay was performed to determine the gene expression of iNOS, IL-6, and TNF-α (Figures 6E-G). After incubation with LFP-80-W1 or LPS for 24 h, the mRNA expression level of iNOS, TNF-α and IL-6 were significantly enhanced and consistent with the result of ELISA assays. The effect of LFP-80-W1 on cytokine release was found to be the most obvious in stimulating IL-6. At the same time, we found that the mRNA expression of iNOS and other cytokines induced by LFP-80-W1 was lower than that of the LPS model control group. Therefore, these findings preliminarily indicated that LFP-80-W1 stimulated the release of NO and inflammatory factors within a specific safe range and did not achieve the effect of causing inflammation.
Effects of LFP-80-W1 on the activation of MAPKs signaling pathway MAPK signaling pathway is a cascade phosphorylation process, which enters the nucleus after activation and regulates transcriptional regulation. The ERK, JNK, and p38 cascade subclasses of MAPK may play an important role in inflammation and apoptosis (33). As shown in Figures 7A,  B, the protein activation of MAPK pathway p-p38, p-ERK, and p-JNK levels were promoted when the cells were treated with 10, 30, and 100 µg/mL of LFP-80-W1 or LPS. Moreover, it was clearly identified that LFP-80-W1 at 100 µg/mL caused the highest phosphorylation levels of ERK, JNK, and p38, similar to those in the LPS group. Therefore, these results indicate that LFP-80-W1 could activate the phosphorylation level of key proteins of the MAPK signaling pathway in RAW 264.7 cells.
No matter what the backbone is composed of, the immunological activity of arabinogalactan depends on the type and the degree of branching (35). The multi-branch structure of polysaccharides is a recognition site for the host complement system and an activator of macrophages (36). During our study, LFP-80-W1 had a backbone of 1, 6-Galp and contained approximately 30% of the branches at the O-3 position. The Araf branches mainly include 1,5linked, 1,3-linked, 1,2,5-linked, and terminal-Araf residues, while the Galp branches mostly contain 1,3-linked, 1,4linked, and terminal-Galp residues. Furthermore, the bioactivity results have indicated that LFP-80-W1 significantly promoted RAW 264.7 cells proliferation and increased the NO, IL-6, and TNF-α cytokines, suggesting the high branching degree and the length of the branched chain of LFP-80-W1 may be its immunostimulatory active site. Notably, in addition to immune activity, specific structural regions also have a significant impact on anti-aging activity. The anti-aging activity of LFPs decreased after the removal of a large number of arabinose side chains by partial acid hydrolysis, suggesting that branching was an essential part of LBPs to exert anti-aging activity (37).

Conclusion
The present work revealed the macromolecular structure of novel arabinogalactan LFP-80-W1 purified from Lycii fructus and preliminarily elucidated the immune regulatory mechanism of RAW 264.7 cells. The dominant LFP-80-W1 was a highly branched heterogeneous comprised of a 1,6linked β-Galp backbone and branched at the O-3 position, with neutral arabinan and arabinogalactan domains. The immunostimulatory effects of exogenous polysaccharide LFP-80-W1 were evaluated at the cellular level. In detail, LFP-80-W1 could dose-dependently increase the intracellular levels of NO, IL-6 and TNF-α and activate the MAPK signaling pathways by upregulating p-ERK, p-JNK, and p-p38. Collectively, these results could contribute to a better understanding of the molecular mechanism of LFP-80-W1 as an effective adjuvant for immunostimulatory activity. Accordingly, Lycii fructus polysaccharides containing LFP-80-W1 may be used to enhance immune function and prevent diseases.

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
XL: experiment design, performing the experiments, writing-original draft, and formal analysis. ML and WC: performing the experiments and formal analysis. SG: experiment design, formal analysis, writing-review and editing, and funding acquisition. FZh: writing original draft, formal analysis, and writing-review and editing. FZe and MX: writing-original draft and review and editing. DQ: writingreview and editing. JD: formal analysis, writing review-editing, and funding acquisition. All authors contributed to the article and approved the submitted version.

Funding
This study was supported by the National Natural Science Foundation of China Regional Innovation and Development Joint Fund Key Project (No. U21A20408), the Key Projects of Key R&D Programs of Ningxia Hui Autonomous Region (Nos. 2021BEF01003, 2021BEF02009, 2021BEF02010, and 2021BEF02012), and the Key Projects of Key R&D Programs of Jiangsu Province (Nos. BE2021907 and BE2021912).