3.2 Structural characterization of the polysaccharides
The contents of total carbohydrate, uronic acid, and protein were determined by colorimetric analyses. As summarized in Table 1, the four polysaccharides contained large amounts of carbohydrates and small amounts of proteins (< 2%). No uronic acid was detected in SFNP-1 and SFNP-2, which suggests that these polysaccharides are neutral polysaccharides. By contrast, large amounts of uronic acids were determined in SFAP-1 and SFAP-2, which suggests their acidic property.
The monosaccharide compositions of the purified polysaccharides were determined as alditol acetates by GC–MS. SFNP-1 and SFNP-2 were mainly composed of glucose, small amounts of arabinose(Ara) and Gal, and small amounts of xylose. Before detecting the monosaccharide composition of the acidic polysaccharides, the -COOH groups of uronic acid residues were first reduced to -CH2OH. It is concluded that the three polysaccharide samples are completely reduced as suggested from the disappearance of the aborsbance band around at 1750 cm− 1 in the FT-IR spectrum(Fig. 3).Monosaccharide composition analysis showed that a large amount of GalA and small amounts of Gal and Ara are present in SFAP-1 and SFAP-2, thus supporting the uronic acid content results. Rhamnose (Rha) was also detected in small amounts (4.6%) in SFAP-2but in trace amounts in SFAP-1. The presence of predominantly composed of GalA residues, with minor amounts of Ara, Gal, and Rha residues, suggesting their pectin-type feature [21]. The GalA glycosyl residues were calculated from the increase in amount of Gal residues in the reduced polysaccharides compared with that in native polysaccharides.
The glycosyl-linkage composition of the two neutral polysaccharides was analyzed by methylation and GC–MS determination. As shown in Fig. 4, (1→4)-linked Glcp residues dominated the glycosyl residues in SFNP-1 and SFNP-2, thus suggesting the presence of (1→4)-linked glucans. Small amounts of terminally linked (t-) and (1→4, 6)-linked Glcp residues were observed, thus suggesting a small amount of branching at the O-4 position of the backbone chain residues. In addition, small amounts of t- and (1→5)-linked Araf and t-, (1→3)-, (1→4)-, and (1→3,6)-linked Galp residues were determined, as summarized in Table 2. These glycosyl residues may reflect the presence of impurities.
Table 2
The linkage analysis of SFNP-1 and SFNP-2 determined by methylation and GC-MS analyses.
Glycosyl residues
|
Substituted position
|
SFNP-1
(mol %)
|
SFNP-2
(mol %)
|
Araf
|
|
|
|
|
T a -
|
3.8
|
3.7
|
|
1,5-
|
9.8
|
5.4
|
Xylp
|
|
|
|
|
1,4-
|
3.1
|
Tr. b
|
Galp
|
|
|
|
|
T-
|
3.4
|
-
|
|
1,3-
|
2.1
|
-
|
|
1,4-
|
2.1
|
2.6
|
|
1,3,6-
|
4.8
|
-
|
Glcp
|
|
|
|
|
T-
|
5.1
|
3.0
|
|
1,4-
|
62.0
|
80.8
|
|
1,4,6-
|
3.7
|
4.6
|
a terminally linked; b trace amount. |
The linkages of GalA residues are usually deduced from increases in Gal residues compared with those in the native form. Therefore, equal amounts of SFAP-1 and SFAP-1R andofSFAP-2 and SFAP-2R were subjected to methylation analysis. When the PMAA derivatives were applied to GC–MS analysis, the PMAAs from native polysaccharides were not detected; this finding maybe due to the predominant presence of GalA, which is resistant to TFA hydrolysis and, therefore, lost during the post-treatment steps, as reported previously [22]. The results suggest that the amount of Gal residues in reduced form could serve as GalA residues in native form SFAP-1 or SFAP-2. According to this deduction, GalA predominantly exists as (1 → 4)-linked GalAp, and small amounts of GalA occur as terminally linked GalA residues (t-GalAp). Small amounts of terminally linked, (1→5)-, and (1→3, 5)-linked arabinosyl residues were also observed. Thus, SFAP-1 and SFAP-2 were deduced to be typical pectin-type polysaccharides containing a homo-galacturonan backbone due to their dominant feature of a linear chain of (1 → 4)-linked GalA units (smooth region)[23].
To further interpretation of the backbone structure of SFAP-1 and SFAP-2, 1D- and 2D-NMR spectra were recorded to examine the backbone structures of SFAP-1 and SFAP-2. As shown in Fig. 5, the 1H- and 13C-NMR spectra of SFAP-1 and SFAP-2 are highly similar, thus suggesting comparable structural characteristics, consistent with the results of methylation analysis. Thus, only the structure of SFAP-1 was analyzed here.
In the 1H-NMR spectrum of SFAP-1 (Fig. 5a), the strong signal at δ3.75 ppm is derived from the esterified methyl groups of GalA, as deduced from the correlation between δ3.75 and δ170.7 ppm in the HMBC spectra and comparison with the reported values [19, 22]. The signal at δ52.8 ppm could be assigned to the esterified methyl groups of GalA, and signals at δ174.6 and δ170.7 ppm were attributed to the carboxyl groups of GalA (residue A) and methyl esterified GalA (residue B), respectively [22]. In the HSQC spectrum, the three signals at δ5.02, δ5.04, and δ4.90 ppm were respectively assigned to H-1 of residues A, B, and C in the 1H-NMR spectrum and corresponded to the carbon signals at δ100.1, δ100.5, and δ101.0 ppm in the 13C-NMR spectrum (Fig. 5b). In the high-field region, four major signals at δ3.67 (67.9), δ4.00 (68.4), δ4.41 (78.5), and δ5.10 (70.5) ppm were respectively assigned to H-2 (C-2), H-3 (C-3), H-4 (C-4), and H-5 (C-5) of the major residue B According to the HSQC spectrum and literature data [22–25], residue A is α-(1→4)-linked D-GalA. The strong signal at δ174.6 ppm, which is assigned to the carboxyl group (C-6) of residue A, supports this deduction. Moreover, according to the HSQC spectrum, among the signals obtained, H-5 appeared to overlap with the HDO signal (Fig. 5c). The anomeric signal at δ4.90 (100.0) ppm and the signal at δ174.6 ppm were assigned to terminally linked D-GalA resulting from residue C on the basis of the HSQC and literature data [22–25]. The chemical shifts of H-2, H-3, H-4, and H-5 of residues A and C were deduced from the HSQC spectrum, as shown in Table 3. Weak signals(δ1.32/19.6 ppm) appearing in the high-field region of the 1H- and 13C-NMR spectra were assigned to H6 and C6 of the Rha residue, thus suggesting the presence of a small amount of Rha. Although the correlation signals of residues A, B, and C were not observed in the HMBC spectrum (not shown here), the results of methylation analysis and the reference NMR data imply that SFAP-1 is a galacturonan predominantly composed of highly methyl-esterified α-(1→4)-linked GalA residues. The degree of methyl-esterification (DM) was estimated to be 57.4% on the basis of the reported method [26]. The weak signal at δ2.02 ppm suggests that the backbone sugar may also be substituted at the O-2 and/or O-3 positions by small amounts of acetyl groups, similar to the structure of previously reported pectin-type polysaccharides [26]. Taking the results together, SFAP-1 was deduced to be a native pectin-type polysaccharide containing a homo-galacturanan backbone consisting of α-(1→4)-linked D-GalAp and methyl-esterified α-(1→4)-linked D-GalAp residues at a ratio ofapproximately1:1.
Table 3
1H-, 13C-NMR chemical shift data (δ ppm) of the major sugar residues in main chain of SFAP-1.
Residues
|
C1/H1
|
C2/H2
|
C3/H3
|
C4/H4
|
C5/H5
|
C6
|
OCH3
|
A a
|
99.1
5.02
|
67.9
3.67
|
68.4
4.00
|
77.9
4.38
|
71.0
4.79
|
174.6
|
|
B b
|
99.6
5.04
|
67.9
3.67
|
68.4
4.00
|
78.5
4.41
|
70.5
5.10
|
170.7
|
52.8
3.75
|
C c
|
100.0
4.90
|
67.9
3.67
|
68.4
4.00
|
79.0
4.64
|
71.0
4.79
|
174.6
|
|
a the residue of (1→4)-α-GalAp; b the residue of methyl-esterified (1→4)-α-GalAp; c the residue of terminally linked α-GalAp. |
Based on its similarity to SFAP-1, SFAP-2 mainly contains a homo-galacturonan composed of highly methyl-esterified and partially acetylated α-(1→4)-linked GalA residues. The DM of this polysaccharide was estimated to be 55.2%.
3.3 Cytotoxicity and anti-inflammatory activities of the polysaccharides
In this experiment, the cytotoxicity of the sugar-containing parts of S. flavescens was tested. As shown in Fig. 6, the crude polysaccharide fraction SFCP and other fractions (SFN and SFA) exhibited cytotoxicity at concentrations higher than 200 µg/mL. After purification, neither SFNP-1 and SFNP-2 isolated from SFN nor SFAP-1 and SFAP-2 isolated from SFA showed any cytotoxicity.
Inflammation is a natural biological response to injury or infection in the human body. Inhibition of the production of inflammatory mediators, such as NO, and inflammatory cytokines, such as tumor necrosis factor α,interleukin 6, and interleukin 1β, serves as a key mechanism in controlling inflammation [27]. In this study, the inflammatory mediator NO was used to test of the anti-inflammation activity of the extracted polysaccharides. LPS, as an endotoxin from Gram-negative bacteria, can induce macrophages to release the inflammatory mediator NO and proinflammatory factors. Therefore, LPS was employed in the present work to stimulate RAW264.7 cells and build an experimentally inflammatory model in vitro.
The inflammatory cell model was constructed by stimulating macrophages with LPS to release NO; then the samples at the concentrations of 100, 500, and 1000 µg/mL were added. Because the significant cell proliferation activity of neutral polysaccharides, here, only acidic polysaccharides, i.e., SFAP-1 and SFAP-2 were applied to evaluate anti-inflammation effects. The results are shown in Fig. 7a.The purified acidic polysaccharides SFAP-1 and SFAP-2 did not present any inhibitory effect on NO release. On the contrary, they significantly stimulated NO production in a dose-dependent manner compared with not only the control group (P < 0.001) but also the LPS group at high-concentrations (100, 500, 1000 µg/mL). Some polysaccharides have been reported to show anti-inflammation activity at low concentrations. For instance, a polysaccharide from Moringa oleifera roots, MRP-1, exhibits anti-inflammation activity by suppressing the release of NO when applied at concentrations of 25, 50, and 100 µg/mL[28]. The Apios americana Medikus tuber polysaccharide ATP-1 suppresses NO release inLPS-induced RAW264.7 cells when applied at concentrations of50, 100, and 150 µg/mL [29]. Thus, the anti-inflammatory activities of low concentrations ofSFAP-1 and SFAP-2 toward NO production in LPS-induced RAW264.7 cells were evaluated. As shown in Fig. 7b, neither of the polysaccharides significantly inhibited NO production compared with the LPS group. In summary, SFAP-1 and SFAP-2 cannot inhibit NO release in LPS-induced RAW264.7 cells regardless of the applied concentration. This finding suggests that SFAP-1 and SFAP-2 do not possess anti-inflammatory activity. Our results demonstrate that the polysaccharides of the decoction of S. flavescens may not be the major effective substances with anti-inflammatory activity.