Structural Features of Three Hetero-Galacturonans from Passiflora foetida Fruits and Their in Vitro Immunomodulatory Effects

Passiflora foetida is a horticultural plant and vital traditional Chinese herbal medicine. In our previous study, the characterization and immuno-enhancing effect of fruits polysaccharide 1 (PFP1), a water-eluted hetero-mannan from wild Passiflora foetida fruits, were investigated. Herein, another three salt-eluted novel polysaccharides, namely PFP2, PFP3, and PFP4, were obtained and structurally characterized. The results showed that PFP2, PFP3, and PFP4 were three structurally similar hetero-galacturonans with different molecular weights of 6.11 × 104, 4.37 × 104, and 3.48 × 105 g/mol, respectively. All three of these hetero-galacturonans are mainly composed of galacturonic acid, galactose, arabinose (75.69%, 80.39%, and 74.30%, respectively), and other monosaccharides including mannose, fucose, glucose, ribose, xylose, and glucuronic acid (24.31%, 19.61, and 25.70%, respectively), although differences in their backbone structure exist. Additionally, immunomodulatory assay indicated that the three hetero-galacturonans possess the ability to promote the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in RAW264.7 macrophages in a concentration-dependent manner (p < 0.05). Especially, PFP3 displayed a stronger enhancing effect than PFP2 and PFP4 at the minimum effective concentration. Therefore, the results suggested that the obtained three salt-eluted hetero-galacturonans, especially PFP3, could be utilized as immunomodulatory effectivity ingredients in nutritional/pharmaceutical industries.


Introduction
Passiflora foetida, known as "Long-zhu" in Chinese, belongs to the genus Passiflora and is mainly distributed in tropical or subtropical areas. Studies have reported that P. foetida fruits possessed a wide range of pharmacological activities, such as antibacterial, anti-osteoporotic, anti-inflammatory, and antioxidant properties [1][2][3][4]. In our previous study, a novel water-eluted hetero-mannan, namely PFP1, was separated from the crude P. foetida fruits polysaccharide (PFP). The hetero-mannan exhibited significant immuno-enhancing effect on RAW264.7 macrophages by promoting the production of NO and tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). However, the obtained PFP1

Separation and Purification of Salt-Eluted Polysaccharides
The crude P. foetida fruits polysaccharide (PFP) was obtained via the same method as in our previous study [13]. Dried PFP was completely dissolved in deionized water (1:20, w/v), then loaded (50 mL) on a DEAE sepharose fast flow column (1.6 cm × 40 cm, Buchi, Flawil, Switzerland) and eluted subsequently with distilled water, 0.1, 0.2, and 0.3 mol/L NaCl solution at a flow rate of 1 mL/min. The total polysaccharide content of eluent (10 mL/tube) was analyzed by the phenol-sulfuric acid method [14]. The peaks were collected, lyophilized, and further purified on a Sephadex G-100 chromatography (1.6 cm × 60 cm, Buchi, Flawil, Switzerland) eluted with 0.1 mol/L NaCl solution at a flow rate of 0.2 mL/min. The purified polysaccharide fractions were obtained after the concentration and lyophilization.

UV-Visible and FT-IR Spectra Analysis
A NanoDrop 2000c UV-Visible spectra (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to check the presence of a nucleic acid-protein of purified polysaccharides solution (50 µg/mL) in the range of 190 to 800 nm. The FT-IR spectra of these samples (KBr pellets) were obtained via a VERTEX 33 FT-IR instrument (Bruker, Rheinstetten, Germany) in the frequency range from 4000 to 400 cm −1 .

Molecular Weight Analysis
The molecular weight distribution of these samples was determined by GPC on a Waters Series 1500 system, equipped with a TSKgel G3000PWXL column (7.8 mm × 300 mm, Tosoh, Japan) and a TSKgel G6000PWXL column (7.8 mm × 300 mm, Tosoh, Japan). The system was connected with a Wyatt MALS detector (DAWN HELEOS II, Wyatt technology, Santa Barbara, CA, USA) and a Wyatt RI detector (Optilab Trex, Wyatt technology, Santa Barbara, CA, USA). Each polysaccharide fraction (10 ± 0.05 mg) was added to 90% dimethylsulfoxide (DMSO, v/v, 1 mL) overnight at 100 • C, followed by centrifugation. Then, the precipitate was washed twice with anhydrous ethanol (3 mL) to remove the residual DMSO. Afterwards, the dried precipitate was dissolved in 0.1 mol/L NaNO 3 (3 mL) at 121 • C for 20 min, and centrifuged (12,000 rpm, 10 min) for analysis. The sample (100 µL) was eluted off by NaNO 3 solution (0.1'mol/L) at a flow rate of 0.4 mL/min and the column oven was kept at 60 • C. The data was analyzed using ASTRA6.1 software (Wyatt Technology Corporation, Santa Barbara, CA, USA).

Molecular Morphological analysis
Dried polysaccharide samples were flimsily plated with platinum and visualized through an Zeiss SEM system (LEO1530VP, Oberkochen, Germany) at a 15 kV accelerating voltage with different image magnifications of 1000×, 3000×, and 5000×, respectively.

Methylation Analysis
The reduction of uronic acids and methylation analysis were carried out as described previously [15,16] with minor modification. Briefly, polysaccharide samples (10 ± 0.05 mg) were reduced with sodium borohydride (NaBH 4 ) and boron sodium deuteride (NaBD 4 ), then dialyzed and lyophilized to acquire the reduzates. Then the reduzates were methylated with DMSO/NaOH and CH 3 I. The FT-IR spectrum was utilized to confirm that the methylations were completed.
The permethylated samples were successively hydrolyzed with 1 mL of 2 M TFA at 121 • C for 1.5 h, then reduced with NaBD 4 and acetylated with acetic anhydride. The eventual derivatives were dissolved in chloroform and injected into a GC-MS analysis on an Agilent 6890A-5975C instrument equipped with Agilent HP-5 capillary column (Santa Clara, CA, USA, 19091S-433, 30.0 m × 0.25 Polymers 2020, 12, 615 4 of 21 mm × 0.25 µm). The column temperature was set to 140 • C during injection, and held for 2 min, then increased by 3 • C/min to 230 • C and maintained at 230 • C for 3 min. Helium was used as the carrier gas. Mass spectra were interpreted to identify the compounds that corresponded to each peak. The molar ratio of each residue was calculated based on peak areas.

NMR Analysis
The linkages and configuration of these purified polysaccharides were identified using 1 H and 13 C NMR spectroscopy. Each polysaccharide fraction (20 ± 0.05 mg) was fully dispersed in ultrapure water (1 mL), centrifuged to remove the precipitation, and the supernatant was lyophilized. The operation was repeated three times, and then the finally lyophilized samples were completely dispersed in 0.5 mL D 2 O, including 1 µL acetone [17]. NMR was recorded on a Bruker AVANCE 600 MHz spectrometer (Billerica, MA, USA). The spectra were observed at 25 • C from 4000 scans.

Immunomodulatory Effect Analysis
RAW264.7 murine macrophage cells (500 µL) were seeded into a 24-well microplate at a density of 1 × 10 5 cells/mL and cultured in DEME culture media (supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin) at 37 • C in 5% CO 2 humidified atmosphere. After cell adhesion had formed or happened for 24 h, the conditioned culture mediums were replaced with 1 mL new DEME culture medium, in which containing different concentrations of PFP2, PFP3, and PFP4 (0.032, 0.16, 0.8, 4, or 20 µg/mL) or culture medium (negative control) for 24 h. In addition, cells cultured in LPS (1 µg/mL) were used as positive control. To exclude the probability of LPS contamination during the polysaccharide isolation period, each polysaccharide fraction sample (20 µg/mL) was blended with various concentrations of polymyxin B (0, 100, or 200 µg/mL) for 0.5 h before added to the medium [10]. The conditioned culture medium was collected and centrifuged (12,000 rpm, 10 min, 4 • C) to determine the content of NO, TNF-α and IL-6 from RAW264.7 cells by using commercial kits in accordance with the manufacturer's instructions. The level of NO in the supernatant was measured using Griess method and was estimated from a sodium nitrite standard curve. Cytokines (TNF-α and IL-6) in the supernatant were measured by ELISA assay according to manufactures' instructions. The absorbance was determined at 540 nm using a microplate reader.

Statistical Analysis
All results were presented as the mean ± standard error of the mean (SEM), and data were analyzed using a one-way analysis of variance. P values less than 0.05 were considered statistically significant.

Extraction and Purification of the Polysaccharide Fractions
The PFP was separated on a DEAE-cellulose Fast Flow column, and a separation flow diagram for PFP was illustrated in Figure 1A. In addition to PFP1, the other three fractions, namely PFP2, PFP3, and PFP4, were obtained according to the order of the elution peaks with the yields of 19.8%, 26.2%, and 12.7%, respectively. These three salt-eluted fractions were further purified by a Sephadex G-100 column. Three single and symmetrically sharp peaks were presented in Figure 1B. Results revealed that PFP2, PFP3, and PFP4 were homogeneous polysaccharides. After purification, PFP2, PFP3, and PFP4, with the total carbohydrate purity of 80.1%, 76.3%, and 78.2%, respectively, were subjected to further chemical analysis.

Analysis of UV-Vis and FT-IR Spectra
UV-Vis spectra ( Figure 1C) showed that PFP2, PFP3, and PFP4 had almost no visual absorption at 260 or 280 nm, suggesting the deficiency of protein or nucleic acids (< 3%) in these polysaccharides.
Like PFP1, the FT-IR spectra of the PFP2, PFP3, and PFP4 fractions showed representative absorbing bands of acidic polysaccharides in the range of 4000-400 cm −1 ( Figure 1D). The vibration regions of PFP2, PFP3, and PFP4 approximately at 3417.75 cm −1 may be due to the stretching vibration of O-H. The broad bands at 2933.63 cm −1 were assigned to the stretching vibration of C-H in the sugar ring [18]. Stretching peaks at 1616.30 cm −1 represented the specific stretching vibration of C=C bonds, as well as at 1424.38 cm −1 , which could be attributed to symmetric COO-, representing the presence of uronic acids [19]. The strong peaks observed at 1000-1200 cm −1 (1039.60 cm −1 ) were assigned to their C-O-H and C-O-C stretching vibrations. The weak absorption bands at around 821.65 cm −1 might be combined with a-pyranose [20]. These results further confirmed that PFP2, PFP3, and PFP4 were acidic polysaccharides.

Morphological Properties
The morphological properties of PFP2, PFP3, and PFP4 through SEM ( Figure 1E-G) were performed at magnifications of 1000×, 3000×, and 5000×. These three polysaccharides were principally formed of various anomalous flakes with increasing degrees of stretch, which might due to the discrepant highly branching construction [21]. Differently, PFP2 had a rough laminated structure with some small distinct pore, while PFP3 and PFP4 had a flake-like structure with a smooth surface. The diversity might be caused by the differences in the water holding capacity and the removal of water during the lyophilization process [22].

Molecular Weight and Monosaccharide Composition
As shown in Table 1, the molecular weights of PFP2, PFP3, and PFP4 were 6.11 × 10 4 g/mol, 4.37 × 10 4 g/mol, and 3.48 × 10 5 g/mol, respectively. Polydispersity index (M w /M n ) of PFP2 (1.58), PFP3 (3.82), and PFP4 (6.18) were lower than that of PFP1 (7.14), which suggested that these three fractions were polydisperse polysaccharides with a lower distribution of molecular weights than that of PFP1 [13]. Combined with the molecular weight of P. edulis polysaccharide (6.0 × 10 4 g/mol) [23], we can see that the polysaccharides extracted from Passiflora spp. fruits possessed relatively high molecular weight. Among these polysaccharide fractions, PFP3 had the lowest molecular weight, and PFP4 had the highest molecular weight. As illustrated in Figure 2, nine monosaccharide compositions were determined by an ion chromatograph. PFP2, PFP3, and PFP4 contained two uronic acids (glucuronic acid and galacturonic acid) and six neutral monosaccharides (mannose, arabinose, fucose, glucose, galactose, and xylose). Traces of ribose was detected only in PFP2. In addition, galacturonic acid, galactose, and arabinose were the main sugar units of these acidic polysaccharides (Table 1), this was completely different from PFP1, which was mainly composed of mannose and galactose. Similar results were observed in P. edulis polysaccharide (PFCM), which was dominated by galacturonic acid (44.2%) [23]. The results shown above speculated that PFP2, PFP3, and PFP4 might have linear galacturonans and heterogeneous sugars branched structures.

NMR Analysis
As illustrated in Figure 3, the 1 H-and 13 C-NMR spectra of these three polysaccharides had similar signal patterns. PFP2, PFP3, and PFP4 had eight similar anomeric proton signals at δ 5. 30 A, B, C, D, E, F, G, and H, respectively. Besides, both PFP2 and PFP4 had two chemical shift signals at δ 5.00/5.01, 4.76/4.74, which were assigned I and J, respectively. PFP2 had a unique residue, appointed as K, in the anomeric proton signals at δ 5.07. Signals of 1, 2-Galp residue were not detected due to low resolution. Signals within the range of 3.32−5.39 ppm ( 1 H NMR) and 59.55−175.20 ppm ( 13 C NMR) represented a typical characteristic of polysaccharides. All the 1 H and 13 C (Table 3) were portioned as thoroughly as possible according to 2D NMR analysis and on literature values [6,10,[24][25][26][27][28][29].
Polymers 2018, 10, x FOR PEER REVIEW 9 of 23 addition to the differences in molar ratios of common residues, the presence or absence of these residues may be the cause of structural differences among PFP2, PFP3, and PFP4.

NMR Analysis
As illustrated in Figure 3, the 1 H-and 13 C-NMR spectra of these three polysaccharides had similar signal patterns. PFP2, PFP3, and PFP4 had eight similar anomeric proton signals at δ 5. 30 A, B, C, D, E, F, G, and H, respectively. Besides, both PFP2 and PFP4 had two chemical shift signals at δ 5.00/5.01, 4.76/4.74, which were assigned I and J, respectively. PFP2 had a unique residue, appointed as K, in the anomeric proton signals at δ 5.07.  Generally, the anomeric signals represent the type of α-configuration in 5.0-5.8 ppm region and represent the β-configuration in 4.4-5.0 ppm region. Thus, residues A, D, E, F, I, and K were α-configuration and residues B, C, G, H, and J were β-configuration. According to the methylation analysis, GalpAc residues were expected to be the most abundant residues. The residues D and G had anomeric signals at δ 5.07/5.02/5.14 and 4.83/4.92/4.97 ppm, while signals at δ 175.2/174.0/173.2 and 103.99/100.96/102.64 ppm in the 13 C NMR indicating that the residues D and G might be galacturonic acid moiety. Based on the proton chemical shifts, 1 H and 13 C chemical shifts indicated by 1 H-1 H correlation spectroscopy (COSY) and heteronuclear single quantum coherence (HSQC) spectra (Figure 3), the chemical shifts of residues D and G consistent nearly with the reported values for a methyl glycoside of 1, 4-linked GalpAc and 1, 3, 4-linked GalpAc. In consideration of the results of FT-IR spectra, methylation analysis and NMR results, it could be deduced that the backbones of PFP2, PFP3, and PFP4 were mainly composed of (1→4)-linked-GalpAc and (1→3, 4)-linked-GalpAc residues [6,10,26,30].
The chemical shifts in 1 H-NMR of PFP2, PFP3, and PFP4 in the anomeric proton of residues A were δ 5.30, 5.27, and 5.28 ppm, respectively, while the matching chemical shifts in 13 C NMR of the anomeric carbon were δ 98.32, 97.82, and 98.26 ppm, respectively. Above all of these results are in accordance with the FT-IR and methylation results, demonstrating that residues A were T-linked Araf [27]. Three signals (residues B) showed up at 4.67, 4.66, and 4.64 ppm, which were assigned to anomeric protons of T-linked Xylp residues of PFP2, PFP3, and PFP4, respectively [25]. All the 1 H and 13 C chemical shifts of residue E assigned by COSY and HSQC in this study were consistent with previous studies [28], and the downfield shift of C-4 (80.87, 80.13, and 80.89 ppm) carbon signals with respect to standard values of methyl glycosides indicated that residues E were (1→4)-linked Manp residues. Meanwhile, (1→6)-linked Glcp (residues I) showed their anomeric chemical shifts at 4.96/4.94 ppm and 104.01/104.05 ppm, respectively, for 1 H and 13 C, which were well matched with the methylation analysis results of PFP2 and PFP4 as shown in Table 2 [27].

Immunomodulatory Effect
Macrophage activation is known as a primary and necessary step for innate immune system stimulation. The cytokines released from activated macrophages, such as NO, TNF-α and IL-6, are important in immune responses [11]. Thus, the immunomodulatory activity of these three hetero-galacturonans on macrophage cells RAW264.7 was studied in vitro. For the release of NO, the results showed that PFP2, PFP3, and PFP4 remarkably promoted the secretion of NO from RAW264.7 cells with significant dosage effects (p < 0.05) in the concentrations of 0.032 to 20 µg/mL. ( Figure 4A). Especially, compared to PFP2 and PFP4, PFP3 possessed better ability to increase NO production at the minimum effective concentration of 0.032 µg/mL (p < 0.05). Under the action of treated maximum concentration (20 µg/mL) of PFP2, PFP3, and PFP4, the NO productions were 43.363 µmol/L, 55.040 µmol/L, and 56.748 µmol/L, respectively, which were significantly lower than that of LPS-treated group (60.608 µmol/L) at the concentration of 1 µg/mL (p < 0.05). Since the overproduction of NO could cause apoptosis in macrophages, the above results suggested that the macrophage activation of these three hetero-galacturonans were more moderate than that of LPS. Meanwhile, it can be seen that the activities of PFP2, PFP3, and PFP4 could not be neutralized by polymyxin B, indicating that they were free of LPS ( Figure 4B). Polymers 2018, 10, x FOR PEER REVIEW 20 of 23  Regarding the secretion of TNF-α and IL-6 in RAW264.7 macrophages, ELISA results indicated that the cellular release of TNF-α ( Figure 4C) was increased by PFP2, PFP3, and PFP4, with a remarkable concentration-dependent manner at the concentrations of 0.032 to 20 µg/mL. Additionally, the PFP3 displayed a stronger ability to enhance the secretion of TNF-α than PFP2 and PFP4 at the minimum effective concentration of 0.032 µg/mL (p < 0.05). Meanwhile, all these three hetero-galacturonans could promote the secretion of IL-6 ( Figure 4D) with a significant concentration-dependent manner at the concentrations of 4 and 20 µg/mL (p < 0.05). These results of the present study were in agreement with the studies on hetero-galacturonans obtained from sweet cherry and stem lettuce [10,11]. Therefore, these results suggested that PFP2, PFP3, and PFP4 may play an immunomodulatory role through promoting the secretion of TNF-α, IL-6, and NO in RAW264.7 macrophages, which are essential for killing pathogens, microorganisms, and coordinating various biological activities as intracellular messenger molecules [5].
Immunomodulatory activity is one of the most important activities of galacturonans, but the structure-function relationship of galacturonans is still not clear. In the previous studies, several hetero-galacturonans obtained from sweet cherry [10], stem lettuce [11], Tanacetum vulgare [32], and elderflowers [33] that consisted of (1→4)-GalpAc backbone remarkably promoted the production of NO and cytokines from macrophages in a dose-dependent manner at concentrations between 25 and 200 µg/mL. It can be inferred the main linkage-type of polysaccharides, especially the (1→4)-GalpAc may be responsible for the macrophages activation. However, the main linkage type is not the only determinant of the immunomodulatory effect of polysaccharides. In this study, the immunomodulatory activity of PFP3 was significantly higher than that of the other two fractions. This phenomenon was in accordance with the study on lettuce polysaccharides (SLP-1 and SLP-2), which reported that SLP-2, with lower molecular weight (44 kDa) and the higher galacturonic acid content (69.5%), had higher macrophages activation effect than SLP-1, with the higher molecular weight (90 kDa) and the lower galacturonic acid content (17.6%) [11]. Taken together, it could be speculated that the relative high galacturonic acid content as well as the relatively low molecular weight of galacturonans may be responsible for the higher immunomodulatory effect. Therefore, although the structure-function relationship of galacturonans is still not explicit, the similarity of structural features like PFP2, PFP3, and PFP4 might be a part of the reasons for their immunomodulatory effect.

Conclusions
In conclusion, three novel salt-eluted polysaccharides, namely PFP2, PFP3, and PFP4, were obtained from the fruits of P. foetida. The structural analysis revealed that PFP2, PFP3, and PFP4 were three hetero-galacturonans with different molecular weights and composed of the same monosaccharide units but with different molar ratios. PFP2, PFP3, and PFP4 had similar glycosidic linkage patterns, which were proven to have a linear homogalacturonan (HG) and →3, 4)-β-D -GalpAc-(1→, with branches at the O-4 position, consisting of Araf, Xylp, Manp, and Galp residues in different molar ratios. In addition, PFP2, PFP3, and PFP4 showed significant immunomodulatory effect, which similar to most previously reported plant galacturonans. These results indicated that PFP2, PFP3, and PFP4 have the potential to be novel immunomodulators for use in pharmaceuticals or functional foods and are worth further research.