Structural Characterization and Anticomplement Activity of an Acidic Heteropolysaccharide from Lysimachia christinae Hance

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Materials
• Preparation of LCP-90 and LCP-90-1
• Structure elucidation of LCP-90-1
• Partial acid hhydrolysis
• Anticomplement activity analysis
• Antioxidant activity assay in vitro
• Binding affinities of LCP-90-1 to complement proteins
• Contributorsʼ Statement
• References

Abstract

A novel acidic heteropolysaccharide (LCP-90-1) was isolated and purified from a traditional “heat-clearing” Chinese medicine, Lysimachia christinae Hance. LCP-90-1 (Mw, 20.65 kDa) was composed of Man, Rha, GlcA, Glc, Gal, and Ara, with relative molar ratios of 1.00: 3.00: 11.62: 1.31: 1.64: 5.24. The backbone consisted of 1,4-α-D-GlcpA, 1,4-α-D-Glcp, 1,4-β-L-Rhap, and 1,3,5-α-L-Araf, with three branches of β-D-Galp-(1 → 4)-β-L-Rhap-(1→, α-L-Araf-(1→ and α-D-Manp-(1→ attached to the C-5 position of 1,3,5-α-L-Araf. LCP-90-1 exhibited potent anticomplement activity (CH₅₀: 135.01 ± 0.68 µg/mL) in vitro, which was significantly enhanced with increased glucuronic acid (GlcA) content in its degradation production (LCP-90-1-A, CH₅₀: 28.26 ± 0.39 µg/mL). However, both LCP-90-1 and LCP90-1-A were inactivated after reduction or complete acid hydrolysis. These observations indicated the important role of GlcA in LCP-90-1 and associated derivatives with respect to anticomplement activity. Similarly, compared with LCP-90-1, the antioxidant activity of LCP-90-1-A was also enhanced. Thus, polysaccharides with a high content of GlcA might be important and effective substances of L. christinae.

Introduction

The complement system is one of the most important immune defense systems in humans, and its proper activation is one of the main effector mechanisms of antibody-mediated immunity [1], [2]. However, excessive activation or inappropriate inhibition of complement can lead to various kinds of diseases, including pneumonia, acute respiratory distress syndrome, rheumatoid arthritis, and system lupus erythematosus [3], [4]. Therefore, inhibition of the excessive activation of the complement system can be exploited to treat inflammatory and autoimmune disorders [5], [6]. Finding the bioactive substances with anticomplement activity is proposed as a potential strategy to develop drugs for immune system diseases. However, most clinical complement inhibitors are synthesized with poor selectivity and induce decreased immunity after long-term use [7]. It has been demonstrated that many natural compounds from “heat-clearing” traditional Chinese medicine showed anticomplement activities [8], [9]. Therefore, it is important to seek effective anticomplement ingredients from traditional Chinese medicine.

As a traditional “heat-clearing” Chinese medicine, Lysimachia christinae Hance (Primulaceae) is commonly used in water decoctions to treat urinary calculi diseases and liver inflammation [10]. It has been reported that the flavonoids, phenolic acids, and crude polysaccharides from the plant exhibited antioxidant and anti-inflammatory activities [11], [12], [13], [14], [15], but no studies have focused on the anticomplement activity of the plant. Our preliminary studies showed that the water extract of L. christinae had much more potent anticomplement activity than the alcohol extract, indicating that the polysaccharides might be the major anticomplement components of L. christinae. Further grading precipitation of the water extract by 75% ethanol and 90% ethanol led to the isolation of two crude polysaccharides, LCP-75 and LCP-90, respectively. The anticomplement activity of LCP-90 (CH₅₀: 45.28 ± 0.34 µg/mL) was better than that of LCP-75 (CH₅₀: 113.64 ± 0.65 µg/mL). Therefore, LCP-90 was taken as the research target to search for anticomplement polysaccharides from L. christinae.

In this study, a novel acidic heteropolysaccharide (LCP-90-1) with anticomplement activity was isolated and purified by DEAE-cellulose chromatography from LCP-90 The current research describes structural characterization in detail and explores the relationship of its glucuronic acid content and the anticomplement and antioxidant activities of the polysaccharide.

Results

The whole herb of L. christinae was degreased with ethanol, followed by water extraction and ethanol precipitation. The protein precipitates were removed by TFA, then followed by dialysis and lyophilization, after which LCP-75 (324.72 g, 2.16% yield) and LCP-90 (55.93 g, 0.37% yield) polysaccharides from L. christinae were obtained successively. The detailed extraction procedures are illustrated in [Fig. 1]. The anticomplement activity of LCP-90 (45.28 ± 0.34 µg/mL) was better when compared with LCP-75 (113.64 ± 0.65 µg/mL). Based on activity-guided statistics, LCP-90 was selected for further investigation.

LCP-90 was isolated and purified on a DEAE-cellulose 52 column, and eluted with 0, 0.1, 0.2, 0.4, 0.8, and 1.6 M NaCl solution. The fractions eluted with 0.1 M NaCl solution were combined, concentrated, dialyzed (cutoff 3000 Da), and lyophilized to obtain 320.24 mg LCP-90-1 ([Fig. 2 a]). The homogeneity of LCP-90-1 was determined by HPGPC-ELSD (high-performance gel permeation chromatography with evaporative light scattering detector) ([Fig. 2 b]) and HPGPC-RID (high-performance gel permeation chromatography with refractive index detector) ([Fig. 2 c]), and its relative molecular weight was 20.65 kDa. Based on the calibration curve, the contents of uronic acid, total carbohydrate, and protein of LCP-90-1 were 47.72 ± 0.05, 92.49 ± 2.31, and 0.34 ± 0.04%, respectively, indicating that LCP-90-1 contained practically no protein.

As shown in [Fig. 3 a, b], when compared with the standards, monosaccharide composition analysis of LCP-90-1 indicated mainly mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), glucose (Glc), galactose (Gal), and arabinose (Ara) at molar ratios of 4.2: 12.6: 48.8: 5.5: 6.9: 22.0 ([Table 1]), thus the relative molar ratio was 1.00: 3.00: 11.62: 1.31: 1.64: 5.24, suggesting that LCP-90-1 was an acidic heteropolysaccharide. The monosaccharide absolute configuration of LCP-90-1 was compared with that of the derivatives of standard monosaccharides (Fig. 1S, Supporting Information) and showed that LCP-90-1 was composed of D-Man, L-Rha, D-GlcA, D-Glc, D-Gal, and L-Ara.

The FT-TR spectrum of LCP-90-1 ([Fig. 3 c]) suggested that a broad band at 3267 cm⁻¹ was attributed to an O-H stretching vibration [8], the signal at 2922 cm⁻¹ was attributed to a C – H stretching vibration [16], the series of absorbance peaks at 1557 cm⁻¹ and 1370 cm⁻¹ was attributed to the asymmetrical stretching vibration of COO- and symmetrical COO- stretching vibrations in the carboxyl groups, thereby indicating the presence of uronic acids [17]. A few absorptions near 1000 ~ 1200 cm⁻¹ were C – O stretching frequencies [18]. Moreover, the peak at 1018 cm⁻¹ indicated the existence of pyranose [19]. The results suggested that LCP-90-1 had typical FT-IR absorption peaks of polysaccharide.

As LCP-90-1 contained GlcA, reduction was required prior to methylation. The results suggested that the reduction product of LCP-90-1 contained no uronic acid, determined by the m-hydroxy biphenyl method. Subsequently, the FT-IR spectrum showed the disappearance of a band in the O-H region (3000 ~ 3500 cm⁻¹), thereby confirming that LCP-90-1-R was fully methylated (Fig. 2S, Supporting Information). The methylated product of LCP-90-1 was hydrolyzed, reduced, and acetylated to obtain its partially methylated alditol acetates (PMAAs). The linkage types and corresponding mass fragmentation patterns of LCP-90-1-R were summarized ([Table 2] and Fig. 3S, Supporting Information) according to the typical fragment ions and literature [19], [20], [21]. For LCP-90-1-R, the main terminal residues contained T-linked Araf (6.2%), T-linked Manp (4.1%), and T-linked Galp (7.0%), and the branched residue was 1,3,5-linked Araf (15.6%). Other residues were disubstituted, including 1,4-linked Glcp (54.2%) and 1,4-linked Rhap (12.9%). Thus, PMAAs consisted of six residues, including T-Araf, 1,4-Rhap, 1,3,5-Araf, T-Galp, T-Manp, and 1,4-Glcp with a relative molar ratio of 1.51: 3.15: 3.80: 1.71: 1.00: 13.22. According to the results of the monosaccharide composition, the repetition unit of LCP-90-1 contained one Glc and twelve GlcA, while for the methylation results of carboxyl reduced product LCP-90-1-R, there were thirteen 1,4-Glcp, indicating that the increased Glc was derived from the GlcA reduction of LCP-90-1. So GlcA was 1,4-linked with a relative molar ratio of about 11.88 (GlcA = 13.22 × [11.62/(1.31 + 11.62)]), indicating that the repetition unit of LCP-90-1 contained twelve 1,4-GlcpA. Similarly, the relative molar ratio of 1,4-Glcp was about 1.34 (Glc = 13.22 × [1.31/(1.31 + 11.62)]), suggesting the repetition unit of LCP-90-1 contained one 1,4-Glcp, which was in line with the result of monosaccharide composition. Therefore, LCP-90-1 contained seven components, including T-Araf, 1,4-Rhap, 1,3,5-Araf, T-Galp, T-Manp, 1,4-GlcpA, and 1,4-Glcp at a relative molar ratio of 1.51: 3.15: 3.80: 1.71: 1.00: 11.88: 1.34, indicating a similar relative molar ratio in monosaccharide composition analysis.

The chemical shifts of sugar residues in LCP-90-1 were assigned by the analysis of ¹H, ¹³C, HSQC, ¹H-¹H COSY, and HMBC NMR spectra ([Fig. 4]), combined with the results of glycosyl composition, methylation analysis, and literature data [16], [17], [18], [19]. The ¹³C NMR spectrum of LCP-90-1 showed complex signals in an anomeric region (95 – 115 ppm) ([Fig. 4 b]), two downfield signals at δ 108.90 and 108.88 ppm could be assigned to 1,3,5-α-L-Araf (A) and T-α-L-Araf (B), respectively [22], [23], and their corresponding anomeric proton chemical shifts (5.30 and 5.13 ppm) were further identified by HSQC spectrum ([Fig. 4 c]) combined with ¹H ([Fig. 4 a]) and ¹H-¹H COSY spectra ([Fig. 4 d]). Similarly, in the regions of 103 – 105 ppm, the signal at δ 104.85 ppm could be attributed to the C-1 of T-β-D-Galp (C), correlated to H-1 of it (δ 4.58 ppm) in the HSQC spectrum [24]. The resonance at δ 102.22 ppm was allocated to the C-1 of 1,4-β-L-Rhap (D), and the corresponding anomeric hydrogen chemical shift was ascribed to 4.70 ppm [25]. The small resonance at δ 100.13 ppm was assigned to C-1 of 1,4-α-D-Glcp (E) due to its low amount and the correlation with anomeric proton resonance at 5.42 ppm in the HSQC spectrum [26]. Signals at δ 99.88 and 97.63 ppm were assigned to C-1 of 1,4-α-D-GlcpA (F) and T-α-D-Manp (G), correlating to H-1 of them (δ 5.35 and 5.01 ppm), respectively [17], [27]. The ¹H and ¹³C chemical shift assignments of LCP-90-1 were defined and are summarized in [Table 3].

We further determined LCP-90-1 linkage sites and sequences among residues by analyzing HMBC spectrum ([Fig. 4 e]) signals. The cross-peaks were designated as follows: H1 (5.30 ppm) of residue A with C-4 (78.77 ppm) of residue F (AH1/FC4) suggested the linkage of residue A with residue F through (1 → 4), that is, 1,3,5-α-L-Araf was attached to the C-4 position of 1,4-α-D-GlcpA. Similarly, the cross-peaks at δ 5.35/82.54 ppm (FH1/AC3) indicated that the linkage of residue F with residue A through (1 → 3); the cross-peaks at δ 5.35/78.77 ppm (FH1/FC4) and 99.88/4.49 ppm (FC1/FH4) indicated that the linkage of residue F with residue F through (1 → 4); the cross-peaks at δ 5.42/78.77 ppm (EH1/FC4) and 100.13/4.49 ppm (EC1/FH4) indicated that the linkage of residue E with residue F through (1 → 4); the cross-peaks at δ 4.70/82.54 ppm (DH1/AC3) and 3.81/102.22 ppm (AH3/DC1) indicated that the linkage of residue D with residue A through (1 → 3); the cross-peaks at δ 99.88/3.73 ppm (FC1/DH4) indicated that the linkage of residue F with residue D through (1 → 4); and the cross-peaks at δ 108.90/3.86 ppm (AC1/EH4) indicated that the linkage of residue A with residue E through (1 → 4). These observations suggested that 1,4-β-L-Rhap was attached to the C-3 position of 1,3,5-α-L-Araf; 1,4-α-D-GlcpA was attached to the C-3 position of 1,3,5-α-L-Araf; 1,4-α-D-GlcpA was attached to the C-4 position of 1,4-β-L-Rhap; 1,4-α-D-GlcpA was attached to the C-4 position of 1,4-α-D-GlcpA with repeating units; and 1,4-α-D-Glcp was attached to the C-4 position of 1,4-α-D-GlcpA, which constituted the backbone of LCP-90-1.

Other cross-peaks were designated as follows: the cross-peaks at δ 5.13/70.68 ppm (BH1/AC5) indicated that the linkage of residue B with residue A through (1 → 5), meaning that T-α-L-Araf was attached to the C-5 position of 1,3,5-α-L-Araf; the cross-peaks at δ 5.01/70.68 ppm (GH1/AC5) and 97.63/3.80 ppm (GC1/AH5) indicated that the linkage of residue G with residue A through (1 → 5), T-α-D-Manp was attached to the C-5 position of 1,3,5-α-L-Araf; the cross-peaks at δ 3.80/102.22 ppm (AH5/DC1) indicated that the linkage of residue D with residue A through (1 → 5), 1,4-β-L-Rhap was attached to the C-5 position of 1,3,5-α-L-Araf; and the cross-peaks at δ 4.58/78.72 ppm (CH1/DC4) and 104.85/3.73 ppm (CC1/DH4) indicated that the linkage of residue C with residue D through (1 → 4), T-β-D-Galp was attached to the C-4 position of 1,4-β-L-Rhap, which indicated the position of the branch chain. Based on these analyses and methylation results, the putative structure of repeating units of LCP-90-1 were hypothesized and are shown in [Fig. 5].

Degraded LCP-90-1 products were mainly divided into LCP-90-1-A (40.8 mg) and LCP-90-1-B (4.3 mg) ([Fig. 3 g]), with relative molecular weight decreasing significantly (LCP-90-1-A: 5.18 kDa; LCP-90-1-B: 0.29 kDa). The homogeneity of LCP-90-1-A was verified by HPGPC-ELSD (Fig. 4S, Supporting Information). The monosaccharide composition of LCP-90-1-A was Rha, GlcA, Glc, and Ara at a molar ratio of 6.5: 60.3: 5.3: 27.9, and LCP-90-1-B was comprised of Man, Rha, Gal, and Ara at a molar ratio of 27.4: 29.6: 9.1: 33.9 ([Fig. 3 e, f] and [Table 1]). It showed that LCP-90-1-A contained more than 50% GlcA and had no branched chain. The monosaccharide composition of LCP-90-1-A (GlcA: Rha: Glc: Ara 1.00: 0.11: 0.09: 0.46) was similar to the backbone of LCP-90-1 (GlcA: Rha: Glc; Ara 1.00: 0.18: 0.11: 0.32), which indicates that LCP-90-1-A may be part of the main chain of native polysaccharide LCP-90-1.

Anticomplement activities of LCP-90-1 and its associated derivatives were detected, and the specific values were recorded ([Table 4]). LCP-90-1 showed weaker anticomplement activity (CH₅₀: 135.01 ± 0.68 µg/mL) when compared with heparin (positive control, CH₅₀: 68.11 ± 0.26 µg/mL), but the activity was significantly enhanced and stronger when compared with heparin after degradation (LCP-90-1-A, CH₅₀: 28.26 ± 0.39 µg/mL). It is notable that LCP-90-1-A with a higher GlcA content was more potent than LCP-90-1, while LCP-90-1-B without GlcA exhibited no anticomplement activity. After reduction, LCP-90-1-R (reduction product of LCP-90-1) and LCP-90-1-AR (reduction product of LCP-90-1-A) no longer had GlcA and anticomplement activity. This suggests that a high content of GlcA played an indispensable role in the anticomplement activity of LCP-90-1 and LCP-90-1-A. In addition, none of LCP-90-1-CH (complete acid hydrolysis of LCP-90-1) and LCP-90-1-AH (hydrolysis of LCP-90-1-A), the hydrolysis of LCP-90-1 and LCP-90-1-A, exhibited anticomplement activity. Furthermore, the monosaccharide GlcA had no anticomplement activity. These results indicate that anticomplement activity was not only associated with GlcA content but was also related to the long chain of the polysaccharide.

The antioxidant activities of LCP-90-1 and its associated degradation products (LCP-90-1-A and LCP-90-1-B, n = 3) were determined in vitro using DPPH (2,2-diphenyl-1-picrylhydrazyl, CAS: 1898 – 66 – 4) radical scavenging, FARP (ferric ion reducing antioxidant power), and ABTS⁺ (ABTS, C₁₈H₂₄N₆O₆S₄, CAS: 30931 – 67 – 0) methods. The values are the mean ± SD. The DPPH radical scavenging abilities of LCP-90-1 and LCP-90-1-A ([Fig. 6 a]) suggested the highest rates at 500 µg/mL (42.41 ± 2.31 and 63.50 ± 3.23%, respectively), while LCP-90-1-B showed almost no activity. The IC₅₀ values of LCP-90-1 and LCP-90-1-A were 1757.79 ± 0.50 and 365.56 ± 0.70 µg/mL, respectively. The ferric ion reducing antioxidant power (FRAP) values of LCP-90-1 ranged from 0.50 ± 0.05 to 1.27 ± 0.07 mM, while LCP-90-1-A values ranged from 0.24 ± 0.06 to 1.36 ± 0.06 mM, whereas LCP-90-1-B showed almost no activity ([Fig. 6 b]). The ABTS⁺ scavenging activities of LCP-90-1and LCP-90-1-A ([Fig. 6 c]) showed the highest rates at 1.0 mg/mL (62.19 ± 3.10 and 71.64 ± 3.50%, respectively), while LCP-90-1-B showed almost no activity. The IC₅₀ values of LCP-90-1 and LCP-90-1-A were 0.89 ± 0.08 and 0.52 ± 0.05 mg/mL, respectively. Therefore, both LCP-90-1 and the degradation product LCP-90-1-A exhibited antioxidant activity, and LCP-90-1-A was stronger than LCP-90-1, whereas the degradation byproduct LCP-90-1-B had no antioxidant activity. This indicates that the antioxidant activity of LCP-90-1 may be related to the GlcA content in the backbone, which was also consistent with the results of anticomplement activity in vitro studies. We hypothesize that the GlcA-rich LCP-90-1 may be an important effective substance of L. christinae.

An SPR (surface plasmon resonance) assay was used to investigate interaction dynamics between LCP-90-1 and complement proteins (C1q, C3, and C5). Binding affinity is highly related to the K_D value, while smaller values are associated with higher interaction affinities [21] at concentrations of 3.125 ~ 400 µg/mL displayed valid binding affinities in a dose-dependent manner with complement proteins immobilized on a CM5 chip [Fig. 6 d] (C1q), [Fig. 6 e] (C3), [Fig. 6 f] (C5)]. The K_D values of LCP-90-1 bound to complement proteins C1q, C3, and C5 were 2.555 × 10⁻⁶, 7.346 × 10⁻⁶ and 1.477 × 10⁻⁵ M, respectively. The results of the SPR experiment suggested that LCP-90-1 had strong affinities with C1q, C3, and C5, while C1q and C3 affinities were significantly stronger when compared with C5. These complement proteins played important roles in the activation of the complement system through the classical pathway, which indicated that LCP-90-1 with anticomplement activity in vitro may be valid for the binding of these complement proteins.

Discussion

In this study, a novel acidic heteropolysaccharide (LCP-90-1, containing 47.72 ± 0.05% GlcA) with anticomplement activity (CH₅₀: 135.01 ± 0.68 µg/mL) was extracted, isolated, and purified from L. christinae based on bioassay-guided fractionation. To explore relationships between the structural characteristics of LCP-90-1 and its GlcA content and anticomplement activity, LCP-90-1 was modified by reduction, degradation, and hydrolysis. After reduction, LCP-90-1-R, without GlcA, completely hemolyzed blood cells, indicating no anticomplement activity. After degradation, GlcA content in LCP-90-1-A (part of the LCP-90-1backbone) exceeded 60%, and its anticomplement activity was significantly enhanced (CH₅₀: 28.26 ± 0.39 µg/mL), while the byproduct LCP-90-1-B, without GlcA, had no anticomplement activity. Therefore, GlcA appeared to have indispensable role in anticomplement activity, consistent with our previous study [8]. Furthermore, LCP-90-1 and LCP-90-1-A showed no anticomplement activity after complete acid hydrolysis, nor did LCP-90-1-AR and the GlcA standard. Thus, a relatively complete backbone with a high content of GlcA may be required for the anticomplement activity of LCP-90-1. Also, the results of the SPR experiment suggested that LCP-90-1 with anticomplement activity in vitro may be related to the valid binding of C1q, C3, and C5 complement proteins, which provides a reference for the future research on the mechanism of anticomplement activity of LCP-90-1.

L. christinae has commonly been taken orally as an aqueous decoction to treat inflammation-related diseases, which suggests that its anticomplement activity in vivo may be achieved by oral administration. It has been reported that Hedyotis diffusa polysaccharide with in vitro anticomplement activity could inhibit LPS-induced overactivation of the complement system in mice by oral administration [9]. An additional study revealed that an intravenous injection of Houttuynia cordata polysaccharide was ineffective, while oral administration exerted the therapeutic effect [28]. Considering the large molecular weight of polysaccharide, oral administration is safe and effective, which provides ideas for the subsequent study of the mechanism of anticomplement activity of LCP-90-1 in vivo through oral administration.

Oxidative stress reflects bodily imbalance between pro-oxidants and antioxidants. More free radicals are quenched by the bodyʼs antioxidant defense system, thereby damaging tissue and generating disease [29]. The most effective and widely used strategy to reduce oxidative stress is exogenous antioxidant supplementation, which is similar to excessive complement activation. In addition, in DPPH radical scavenging and FARP and ABTS⁺ assays, LCP-90-1-A showed stronger activity when compared with LCP-90-1, while LCP-90-1-B remained inactive, similar to anticomplement activity. It was previously reported that changing fermentation methods to increase the content of GlcA in apple cider significantly enhanced antioxidant activity [30]. It indicated that increasing the content of GlcA in polysaccharides may enhance both anticomplement and antioxidant activities and provide ideas for the subsequent study of anticomplement and antioxidant drugs.

In conclusion, a novel acidic heteropolysaccharide LCP-90-1 containing a high content of GlcA with anticomplement and antioxidant activities was obtained from L. christinae. The backbone structure mainly formed by GlcA was an important characteristic of the anticomplement and antioxidant polysaccharides of L. christinae. In our study, LCP-90-1 exhibited good development prospects as a potential complement inhibitor and antioxidant and provides a reference point for the structure-activity relationships for polysaccharides with anticomplement and antioxidant activities potential.

Materials and Methods

Materials

The whole herb of L. christinae was purchased from Bozhou Medicinal Materials Market (Anhui Province) and positively identified by Professor Daofeng Chen of Fudan University. The specimen (DFC-LC20200710) was deposited in the School of Pharmacy, Fudan University.

Preparation of LCP-90 and LCP-90-1

The crude polysaccharide of LCP-90 was extracted according to a reported method [8] (for details, see Supporting Information). A detailed extraction procedure is illustrated in [Fig. 1]. A purified polysaccharide, LCP-90-1 (320.24 mg), was isolated and purified from the crude polysaccharide LCP-90 (50 g) (for details, see Supporting Information).

Structure elucidation of LCP-90-1

The homogeneity and relative molecular weight of LCP-90-1 were analyzed by an HPGPC method [31] (for details, see Supporting Information). The relative molecular weight of LCP-90-1 was determined based on the standard curve [32]. The uronic acid content was determined using the m-hydroxy biphenyl method [33] with glucuronic acid (GlcA) as the standard. The total protein content was estimated by the Bradfordʼs method [33] using bovine serum albumin as the standard, and the total carbohydrate content was determined by a phenol-sulfuric acid method using mixed standards as standards based on the results of the monosaccharide composition [33].

The monosaccharide composition of LCP-90-1 was determined by classical PMP (1-phenyl-3 methyl-5-pyrazolone) pre-column derivatization and HPLC analysis [34] (for details, see Supporting Information). The monosaccharide absolute configuration of LCP-90-1 was determined according to the literature [35] (for details, see Supporting Information).

The organic functional groups of the sample (LCP-90-1 and derivatives) was detected by an FT-IR spectrophotometer (Bruker) [36] (for details, see Supporting Information). LCP-90-1 contained uronic acid. A reduction reaction was required prior to methylation [37] (for the method of reduction details, see Supporting Information). The methylation method was based on the method of Hakomori [38], which is detailed in the Supporting Information. The PMAAs of LCP-90-1 were analyzed by GC-MS. The PMAAs connection modes were identified by comparing characteristic fragmentation patterns according to the literature [39]. NMR spectra analysis of LCP-90-1 was performed according to previously published methods [40] in the laboratory (for details, see Supporting Information).

Partial acid hhydrolysis

For hydrolysis, LCP-90-1 (100 mg) was redissolved in 40 mL 1 M TFA solution at 100 °C for 1 h [34] (for details, see Supporting Information). According to the results of HPGPC-ELSD, the residues were separated by HPLC (LCP-90-1-A and LCP-90-1-B), and the monosaccharide composition and anticomplement activity were analyzed.

Anticomplement activity analysis

Anticomplement activity through the classical pathway of samples was analyzed according to our previous work [8] (for details, see Supporting Information). All animal experiment protocols were approved by the Animal Ethical Committee of School of Pharmacy, Fudan University on March 1, 2018 (2018-03-SY-CDF-01) and conducted following the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health. Minimization of animal suffering was ensured during experimental procedures.

Antioxidant activity assay in vitro

The antioxidant activities were determined by DPPH, an ABTS method (ABTS, C₁₈H₂₄N₆O₆S₄, CAS: 30931 – 67 – 0), and FRAP. The DPPH radical scavenging abilities of different LCP-90-1 concentrations (1 ~ 500 µg/mL) and associated degradation products were determined by a method [41], with minor adjustments. In addition, the ABTS⁺ and FRAP radical scavenging ability of different LCP-90-1 concentrations (0.125 ~ 1.5 mg/mL) and associated degradation products were determined using Beyotime enzyme-linked immunosorbent assay kit instructions.

Binding affinities of LCP-90-1 to complement proteins

Complement proteins C1q, C3, and C5 played an important role in the activation of the complement system through the classical pathway [42]. Therefore, based on the principle of SPR, the binding affinities of LCP-90-1 to complement proteins were analyzed by the Molecular Interaction Analysis System (Biacore T200) (for details, see Supporting Information).

Contributorsʼ Statement

Conception and design of the work: Z. Hong, D. F. Chen, Y. Lu; data collection: Z. Hong; analysis and interpretation of the data: Z. Hong, L. S. Zhou, Z. Z. Zhao, G. Q. Yuan, and X. J. Wang; drafting the manuscript: Z. Hong; critical revision of the manuscript: L. S. Zhou, D. F. Chen, Y. Lu.

Conflict of Interest

The authors declare that they have no conflict of interest.

• References

• 1 Braunstein JS, Kirschfink M. Complement deficiencies and dysregulation: Pathophysiological consequences, modern analysis, and clinical management. Mol Immunol 2019; 114: 99-311
  PubMedSearch in Google Scholar
• 2 Garred P, Tenner AJ, Mollnes TE. Therapeutic targeting of the complement system: From rare diseases to pandemics. Pharmacol Rev 2021; 73: 792-827
  CrossrefPubMedSearch in Google Scholar
• 3 Chen M, Daha MR, Kallenberg C. The complement system in systemic autoimmune disease. J Autoimmun 2009; 34: 276-286
  CrossrefPubMedSearch in Google Scholar
• 4 Harboe M, Thorgersen EB, Mollnes TE. Advances in assay of complement function and activation. Adv Drug Deliv Rev 2011; 63: 976-987
  CrossrefPubMedSearch in Google Scholar
• 5 Zoltán P, Michael K, Ashley FA. Complement analysis in the era of targeted therapeutics. Mol Immunol 2018; 102: 84-88
  CrossrefPubMedSearch in Google Scholar
• 6 Stephen T, Thurman JM. Tissue-targeted complement therapeutics. Mol Immunol 2018; 102: 120-128
  CrossrefPubMedSearch in Google Scholar
• 7 Deng Z, Tu W, Deng Z, Hu QN. PhID: An open-access integrated pharmacology interactions database for drugs, targets, diseases, genes, side-effects and pathways. J Chem Inf Model 2017; 57: 2395-2400
  CrossrefPubMedSearch in Google Scholar
• 8 Xia L, De J, Zhu MX, Chen DF, Lu Y. Juniperus pingii var. wilsonii acidic polysaccharide: Extraction, characterization and anticomplement activity. Carbohydr Polym 2020; 231: 115728
  CrossrefPubMedSearch in Google Scholar
• 9 Zhang J, Huo J, Zhao Z, Lu Y, Hong Z, Li H, Chen D. An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation. Phytomedicine 2022; 107: 154453
  CrossrefPubMedSearch in Google Scholar
• 10 China Pharmacopoeia Committee. Pharmacopoeia of the Peopleʼs Republic China, 1st Division of 2010 Edition. Beijing: China Medical Science Press; 2010: 204-205
  Search in Google Scholar
• 11 Zhang FX, Liu XF, Ke ZQ, Wu NH, Chen HG, Liu C. The effects of Lysimachia christinae Hance extract fractions on endothelium-dependent vasodilatation. Pharmacology 2019; 104: 36-42
  CrossrefPubMedSearch in Google Scholar
• 12 Kim AR, Jung MC, Jeong HI, Song DG, Seo YB, Jeon YH, Park SH, Shin HS, Lee SL, Park SN. Antioxidative and cellular protective effects of Lysimachia christinae Hance extract and fractions. BMC Complement Altern Med 2018; 1: 128
  PubMedSearch in Google Scholar
• 13 Wang J, Chen J, Huang J, Lv B, Tao T. Protective effects of total flavonoids from Lysimachia Christinae on calcium oxalate-induced oxidative stress in a renal cell line and renal tissue. Evid Based Complement Alternat Med 2021; 2021: 6667902
  PubMedSearch in Google Scholar
• 14 Wang JM, Zhang YY, Zhang YS, Cui Y, Liu J, Zhang BF. Protective effect of Lysimachia christinae against acute alcohol-induced liver injury in mice. Biosci Trends 2012; 6: 89-97
  PubMedSearch in Google Scholar
• 15 Wang JM, Liu J, Cui Y, Zhang HW, Feng ZY. Hepatoprotective effects of Lysimachia christinae Hance extracts on acute liver injury induced by tripterygium glycosides in mice. Can J Physiol Pharmacol 2013; 93: 427-433
  PubMedSearch in Google Scholar
• 16 Tian YT, Zhao YT, Zeng HL, Zhang YL, Zheng BD. Structural characterization of a novel neutral polysaccharide from Lentinus giganteus and its antitumor activity through inducing apoptosis. Carbohydr Polym 2016; 154: 231-240
  CrossrefPubMedSearch in Google Scholar
• 17 Song Y, Zhu M, Hao H, Deng J, Li M, Sun Y, Yang R, Wang H, Huang R. Structure characterization of a novel polysaccharide from Chinese wild fruits (Passiflora foetida) and its immune-enhancing activity. Int J Biol Macromol 2019; 136: 324-331
  CrossrefPubMedSearch in Google Scholar
• 18 Wang J, Han J, Lu Z, Lu F. Preliminary structure, antioxidant and immunostimulatory activities of a polysaccharide fraction from Artemisia selengensis Turcz. Int J Biol Macromol 2020; 143: 842-849
  CrossrefPubMedSearch in Google Scholar
• 19 Zhou LS, Jiao YK, Tang JY, Zhao ZZ, Zhu HY, Lu Y, Chen DF. Ultrafiltration isolation, structure and effects on H1N1-induced acute lung injury of a heteropolysaccharide from Houttuynia cordata . Int J Biol Macromol 2022; 222: 2414-2425
  CrossrefPubMedSearch in Google Scholar
• 20 Sims I, Carnachan S, Bell T, Hinkley S. Methylation analysis of polysaccharides: Technical advice. Carbohydr Polym 2018; 188: 1-7
  CrossrefPubMedSearch in Google Scholar
• 21 Shen Y, Liang J, Guo YL, Li Y, Kuang HX, Xia YG. Ultrafiltration isolation, structures and anti-tumor potentials of two arabinose- and galactose-rich pectins from leaves of Aralia elata . Carbohydr Polym 2021; 255: 117326
  CrossrefPubMedSearch in Google Scholar
• 22 Qin L, He MJ, Yang YJ, Fu ZT, Mao WJ. Anticoagulant-active sulfated arabinogalactan from Chaetomorpha linum: Structural characterization and action on coagulation factors. Carbohydr Polym 2020; 242: 116394
  CrossrefPubMedSearch in Google Scholar
• 23 Zhang JJ, Song ZT, Li Y, Zhang SJ, Guo YQ. Structural analysis and biological effects of a neutral polysaccharide from the fruits of Rosa laevigata . Carbohydr Polym 2021; 265: 118080
  CrossrefPubMedSearch in Google Scholar
• 24 Bhanja SK, Maity P, Rout D, Sen IK, Patra S. A xylan from the fresh leaves of Piper betle: Structural characterization and studies of bioactive properties. Carbohydr Polym 2022; 291: 119570
  CrossrefPubMedSearch in Google Scholar
• 25 Ganaie F, Maruhn K, Li CX, Porambo RJ, Elverdal PL, Abeygunwardana C, Linden M, Duus J, Sheppard CL, Nahm MH. Structural, Genetic, and Serological Elucidation of Streptococcus pneumoniae Serogroup 24 Serotypes: Discovery of a New Serotype, 24C, with a Variable Capsule Structure. J Clin Microbiol 2021; 59: e0054021
  CrossrefPubMedSearch in Google Scholar
• 26 Zhou Y, Ma W, Wang L, Sun W, Li M, Zhang W, Liu Y, Song X, Fan Y. Characterization and antioxidant activity of the oligo-maltose fraction from Polygonum Cillinerve . Carbohydr Polym 2019; 226: 115307
  CrossrefPubMedSearch in Google Scholar
• 27 Xiong Q, Luo G, Zheng F, Wu K, Yang H, Chen L, Tian W. Structural characterization and evaluation the elicitors activity of polysaccharides from Chrysanthemum indicum . Carbohydr Polym 2021; 263: 117994
  CrossrefPubMedSearch in Google Scholar
• 28 Shi C, Zhu H, Li H, Zeng D, Shi X, Zhang Y, Lu Y, Ling L, Wang C, Chen D. Regulating the balance of Th17/Treg cells in gut-lung axis contributed to the therapeutic effect of Houttuynia cordata polysaccharides on H1N1-induced acute lung injury. Int J Biol Macromol 2020; 158: 52-66
  CrossrefPubMedSearch in Google Scholar
• 29 Zhong Q, Wei B, Wang S, Ke S, Chen J, Zhang H, Wang H. The antioxidant activity of polysaccharides derived from marine organisms: An overview. Mar Drugs 2019; 17: 674
  CrossrefPubMedSearch in Google Scholar
• 30 Li Y, Nguyen T, Jin J, Lim J, Lee J, Piao M, Mok I, Kim D. Brewing of glucuronic acid-enriched apple cider with enhanced antioxidant activities through the co-fermentation of yeast (Saccharomyces cerevisiae and Pichia kudriavzevii) and bacteria (Lactobacillus plantarum). Food Sci Biotechnol 2021; 30: 555-564
  CrossrefPubMedSearch in Google Scholar
• 31 Yue H, Liu Y, Qu H, Ding K. Structure analysis of a novel heteroxylan from the stem of Dendrobium officinale and anti-angiogenesis activities of its sulfated derivative. Int J Biol Macromol 2017; 103: 533-542
  CrossrefPubMedSearch in Google Scholar
• 32 Lin Z, Li T, Yu Q, Chen H, Yan C. Structural characterization and in vitro osteogenic activity of ABPB-4, a heteropolysaccharide from the rhizome of Achyranthes bidentate . Carbohydr Polym 2021; 259: 117553
  CrossrefPubMedSearch in Google Scholar
• 33 Huo JY, Lu Y, Xia L, Chen DF. Structural characterization and anticomplement activities of three acidic homogeneous polysaccharides from Artemisia annua . J Ethnopharmacol 2020; 247: 112281
  CrossrefPubMedSearch in Google Scholar
• 34 Zhou LS, Liao WF, Zeng H, Yao YL, Chen X, Ding K. A pectin from fruits of Lycium barbarum L. decreases β-amyloid peptide production through modulating APP processing. Carbohydr Polym 2018; 201: 65-74
  CrossrefPubMedSearch in Google Scholar
• 35 Chen X, Li T, Qing D, Chen J, Zhang Q, Yan C. Structural characterization and osteogenic bioactivities of a novel Humulus lupulus polysaccharide. Food Funct 2020; 11: 1165-1175
  CrossrefPubMedSearch in Google Scholar
• 36 Zhang H, Zhong J, Zhang Q, Qing DG, Yan CY. Structural elucidation and bioactivities of a novel arabinogalactan from Coreopsis tinctoria . Carbohydr Polym 2019; 219: 219-228
  CrossrefPubMedSearch in Google Scholar
• 37 Li SJ, Li MX, Yue H, Zhou LS, Huang LL, Du ZY, Ding K. Structural elucidation of a pectic polysaccharide from Fructus Mori and its bioactivity on intestinal bacteria strains. Carbohydr Polym 2018; 186: 168-175
  CrossrefPubMedSearch in Google Scholar
• 38 Zhang SJ, Li ZG, Wang XL, An LJ, Bao JH, Guo YQ. Isolation, structural elucidation, and immunoregulation properties of an arabinofuranan from the rinds of Garcinia mangostana . Carbohydr Polym 2020; 246: 116567
  CrossrefPubMedSearch in Google Scholar
• 39 Shi ZY, An LJ, Zhang SJ, Li ZG, Guo YQ. A heteropolysaccharide purified from leaves of Ilex latifolia displaying immunomodulatory activity in vitro and in vivo . Carbohydr Polym 2020; 245: 116469
  CrossrefPubMedSearch in Google Scholar
• 40 Ye ZQ, Li TY, Qing DG, Sun Y, Yan CY. Structural elucidation and osteogenic activity of a novel heteropolysaccharide from Alhagi pseudalhagi . Int J Biol Macromol 2021; 171: 185-197
  CrossrefPubMedSearch in Google Scholar
• 41 Jiang P, Meng W, Shi FX, Chen C, Sun Y, Jiao LL. Structural characteristics, antioxidant properties and antiaging activities of galactan produced by Mentha haplocalyx Briq. Carbohydr Polym 2020; 234: 115936
  CrossrefPubMedSearch in Google Scholar
• 42 Schartz ND, Tenner AJ. The good, the bad, and the opportunities of the complement system in neurodegenerative disease. J Neuroinflammation 2020; 17: 354
  CrossrefPubMedSearch in Google Scholar

Correspondence

Publication History

Received: 27 April 2023

Accepted after revision: 04 August 2023

Accepted Manuscript online:
04 August 2023

Article published online:
23 August 2023

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Braunstein JS, Kirschfink M. Complement deficiencies and dysregulation: Pathophysiological consequences, modern analysis, and clinical management. Mol Immunol 2019; 114: 99-311
  PubMedSearch in Google Scholar
• 2 Garred P, Tenner AJ, Mollnes TE. Therapeutic targeting of the complement system: From rare diseases to pandemics. Pharmacol Rev 2021; 73: 792-827
  CrossrefPubMedSearch in Google Scholar
• 3 Chen M, Daha MR, Kallenberg C. The complement system in systemic autoimmune disease. J Autoimmun 2009; 34: 276-286
  CrossrefPubMedSearch in Google Scholar
• 4 Harboe M, Thorgersen EB, Mollnes TE. Advances in assay of complement function and activation. Adv Drug Deliv Rev 2011; 63: 976-987
  CrossrefPubMedSearch in Google Scholar
• 5 Zoltán P, Michael K, Ashley FA. Complement analysis in the era of targeted therapeutics. Mol Immunol 2018; 102: 84-88
  CrossrefPubMedSearch in Google Scholar
• 6 Stephen T, Thurman JM. Tissue-targeted complement therapeutics. Mol Immunol 2018; 102: 120-128
  CrossrefPubMedSearch in Google Scholar
• 7 Deng Z, Tu W, Deng Z, Hu QN. PhID: An open-access integrated pharmacology interactions database for drugs, targets, diseases, genes, side-effects and pathways. J Chem Inf Model 2017; 57: 2395-2400
  CrossrefPubMedSearch in Google Scholar
• 8 Xia L, De J, Zhu MX, Chen DF, Lu Y. Juniperus pingii var. wilsonii acidic polysaccharide: Extraction, characterization and anticomplement activity. Carbohydr Polym 2020; 231: 115728
  CrossrefPubMedSearch in Google Scholar
• 9 Zhang J, Huo J, Zhao Z, Lu Y, Hong Z, Li H, Chen D. An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation. Phytomedicine 2022; 107: 154453
  CrossrefPubMedSearch in Google Scholar
• 10 China Pharmacopoeia Committee. Pharmacopoeia of the Peopleʼs Republic China, 1st Division of 2010 Edition. Beijing: China Medical Science Press; 2010: 204-205
  Search in Google Scholar
• 11 Zhang FX, Liu XF, Ke ZQ, Wu NH, Chen HG, Liu C. The effects of Lysimachia christinae Hance extract fractions on endothelium-dependent vasodilatation. Pharmacology 2019; 104: 36-42
  CrossrefPubMedSearch in Google Scholar
• 12 Kim AR, Jung MC, Jeong HI, Song DG, Seo YB, Jeon YH, Park SH, Shin HS, Lee SL, Park SN. Antioxidative and cellular protective effects of Lysimachia christinae Hance extract and fractions. BMC Complement Altern Med 2018; 1: 128
  PubMedSearch in Google Scholar
• 13 Wang J, Chen J, Huang J, Lv B, Tao T. Protective effects of total flavonoids from Lysimachia Christinae on calcium oxalate-induced oxidative stress in a renal cell line and renal tissue. Evid Based Complement Alternat Med 2021; 2021: 6667902
  PubMedSearch in Google Scholar
• 14 Wang JM, Zhang YY, Zhang YS, Cui Y, Liu J, Zhang BF. Protective effect of Lysimachia christinae against acute alcohol-induced liver injury in mice. Biosci Trends 2012; 6: 89-97
  PubMedSearch in Google Scholar
• 15 Wang JM, Liu J, Cui Y, Zhang HW, Feng ZY. Hepatoprotective effects of Lysimachia christinae Hance extracts on acute liver injury induced by tripterygium glycosides in mice. Can J Physiol Pharmacol 2013; 93: 427-433
  PubMedSearch in Google Scholar
• 16 Tian YT, Zhao YT, Zeng HL, Zhang YL, Zheng BD. Structural characterization of a novel neutral polysaccharide from Lentinus giganteus and its antitumor activity through inducing apoptosis. Carbohydr Polym 2016; 154: 231-240
  CrossrefPubMedSearch in Google Scholar
• 17 Song Y, Zhu M, Hao H, Deng J, Li M, Sun Y, Yang R, Wang H, Huang R. Structure characterization of a novel polysaccharide from Chinese wild fruits (Passiflora foetida) and its immune-enhancing activity. Int J Biol Macromol 2019; 136: 324-331
  CrossrefPubMedSearch in Google Scholar
• 18 Wang J, Han J, Lu Z, Lu F. Preliminary structure, antioxidant and immunostimulatory activities of a polysaccharide fraction from Artemisia selengensis Turcz. Int J Biol Macromol 2020; 143: 842-849
  CrossrefPubMedSearch in Google Scholar
• 19 Zhou LS, Jiao YK, Tang JY, Zhao ZZ, Zhu HY, Lu Y, Chen DF. Ultrafiltration isolation, structure and effects on H1N1-induced acute lung injury of a heteropolysaccharide from Houttuynia cordata . Int J Biol Macromol 2022; 222: 2414-2425
  CrossrefPubMedSearch in Google Scholar
• 20 Sims I, Carnachan S, Bell T, Hinkley S. Methylation analysis of polysaccharides: Technical advice. Carbohydr Polym 2018; 188: 1-7
  CrossrefPubMedSearch in Google Scholar
• 21 Shen Y, Liang J, Guo YL, Li Y, Kuang HX, Xia YG. Ultrafiltration isolation, structures and anti-tumor potentials of two arabinose- and galactose-rich pectins from leaves of Aralia elata . Carbohydr Polym 2021; 255: 117326
  CrossrefPubMedSearch in Google Scholar
• 22 Qin L, He MJ, Yang YJ, Fu ZT, Mao WJ. Anticoagulant-active sulfated arabinogalactan from Chaetomorpha linum: Structural characterization and action on coagulation factors. Carbohydr Polym 2020; 242: 116394
  CrossrefPubMedSearch in Google Scholar
• 23 Zhang JJ, Song ZT, Li Y, Zhang SJ, Guo YQ. Structural analysis and biological effects of a neutral polysaccharide from the fruits of Rosa laevigata . Carbohydr Polym 2021; 265: 118080
  CrossrefPubMedSearch in Google Scholar
• 24 Bhanja SK, Maity P, Rout D, Sen IK, Patra S. A xylan from the fresh leaves of Piper betle: Structural characterization and studies of bioactive properties. Carbohydr Polym 2022; 291: 119570
  CrossrefPubMedSearch in Google Scholar
• 25 Ganaie F, Maruhn K, Li CX, Porambo RJ, Elverdal PL, Abeygunwardana C, Linden M, Duus J, Sheppard CL, Nahm MH. Structural, Genetic, and Serological Elucidation of Streptococcus pneumoniae Serogroup 24 Serotypes: Discovery of a New Serotype, 24C, with a Variable Capsule Structure. J Clin Microbiol 2021; 59: e0054021
  CrossrefPubMedSearch in Google Scholar
• 26 Zhou Y, Ma W, Wang L, Sun W, Li M, Zhang W, Liu Y, Song X, Fan Y. Characterization and antioxidant activity of the oligo-maltose fraction from Polygonum Cillinerve . Carbohydr Polym 2019; 226: 115307
  CrossrefPubMedSearch in Google Scholar
• 27 Xiong Q, Luo G, Zheng F, Wu K, Yang H, Chen L, Tian W. Structural characterization and evaluation the elicitors activity of polysaccharides from Chrysanthemum indicum . Carbohydr Polym 2021; 263: 117994
  CrossrefPubMedSearch in Google Scholar
• 28 Shi C, Zhu H, Li H, Zeng D, Shi X, Zhang Y, Lu Y, Ling L, Wang C, Chen D. Regulating the balance of Th17/Treg cells in gut-lung axis contributed to the therapeutic effect of Houttuynia cordata polysaccharides on H1N1-induced acute lung injury. Int J Biol Macromol 2020; 158: 52-66
  CrossrefPubMedSearch in Google Scholar
• 29 Zhong Q, Wei B, Wang S, Ke S, Chen J, Zhang H, Wang H. The antioxidant activity of polysaccharides derived from marine organisms: An overview. Mar Drugs 2019; 17: 674
  CrossrefPubMedSearch in Google Scholar
• 30 Li Y, Nguyen T, Jin J, Lim J, Lee J, Piao M, Mok I, Kim D. Brewing of glucuronic acid-enriched apple cider with enhanced antioxidant activities through the co-fermentation of yeast (Saccharomyces cerevisiae and Pichia kudriavzevii) and bacteria (Lactobacillus plantarum). Food Sci Biotechnol 2021; 30: 555-564
  CrossrefPubMedSearch in Google Scholar
• 31 Yue H, Liu Y, Qu H, Ding K. Structure analysis of a novel heteroxylan from the stem of Dendrobium officinale and anti-angiogenesis activities of its sulfated derivative. Int J Biol Macromol 2017; 103: 533-542
  CrossrefPubMedSearch in Google Scholar
• 32 Lin Z, Li T, Yu Q, Chen H, Yan C. Structural characterization and in vitro osteogenic activity of ABPB-4, a heteropolysaccharide from the rhizome of Achyranthes bidentate . Carbohydr Polym 2021; 259: 117553
  CrossrefPubMedSearch in Google Scholar
• 33 Huo JY, Lu Y, Xia L, Chen DF. Structural characterization and anticomplement activities of three acidic homogeneous polysaccharides from Artemisia annua . J Ethnopharmacol 2020; 247: 112281
  CrossrefPubMedSearch in Google Scholar
• 34 Zhou LS, Liao WF, Zeng H, Yao YL, Chen X, Ding K. A pectin from fruits of Lycium barbarum L. decreases β-amyloid peptide production through modulating APP processing. Carbohydr Polym 2018; 201: 65-74
  CrossrefPubMedSearch in Google Scholar
• 35 Chen X, Li T, Qing D, Chen J, Zhang Q, Yan C. Structural characterization and osteogenic bioactivities of a novel Humulus lupulus polysaccharide. Food Funct 2020; 11: 1165-1175
  CrossrefPubMedSearch in Google Scholar
• 36 Zhang H, Zhong J, Zhang Q, Qing DG, Yan CY. Structural elucidation and bioactivities of a novel arabinogalactan from Coreopsis tinctoria . Carbohydr Polym 2019; 219: 219-228
  CrossrefPubMedSearch in Google Scholar
• 37 Li SJ, Li MX, Yue H, Zhou LS, Huang LL, Du ZY, Ding K. Structural elucidation of a pectic polysaccharide from Fructus Mori and its bioactivity on intestinal bacteria strains. Carbohydr Polym 2018; 186: 168-175
  CrossrefPubMedSearch in Google Scholar
• 38 Zhang SJ, Li ZG, Wang XL, An LJ, Bao JH, Guo YQ. Isolation, structural elucidation, and immunoregulation properties of an arabinofuranan from the rinds of Garcinia mangostana . Carbohydr Polym 2020; 246: 116567
  CrossrefPubMedSearch in Google Scholar
• 39 Shi ZY, An LJ, Zhang SJ, Li ZG, Guo YQ. A heteropolysaccharide purified from leaves of Ilex latifolia displaying immunomodulatory activity in vitro and in vivo . Carbohydr Polym 2020; 245: 116469
  CrossrefPubMedSearch in Google Scholar
• 40 Ye ZQ, Li TY, Qing DG, Sun Y, Yan CY. Structural elucidation and osteogenic activity of a novel heteropolysaccharide from Alhagi pseudalhagi . Int J Biol Macromol 2021; 171: 185-197
  CrossrefPubMedSearch in Google Scholar
• 41 Jiang P, Meng W, Shi FX, Chen C, Sun Y, Jiao LL. Structural characteristics, antioxidant properties and antiaging activities of galactan produced by Mentha haplocalyx Briq. Carbohydr Polym 2020; 234: 115936
  CrossrefPubMedSearch in Google Scholar
• 42 Schartz ND, Tenner AJ. The good, the bad, and the opportunities of the complement system in neurodegenerative disease. J Neuroinflammation 2020; 17: 354
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material