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

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-ß-L-Rhap, and 1,3,5-α-L-Araf, with three branches of ß-D-Galp-(1 → 4)-ß-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.

Structural Characterization and Anti-inflammatory Activities of Anticomplementary Polysaccharides from Rhododendron principis.

Rhododendron principis leaves have been used as "Dama", a Traditional Tibetan Medicine for treating inflammatory diseases. R. principis crude polysaccharides with anticomplementary activity demonstrated promising anti-inflammatory effects on acute lung injury induced by lipopolysaccharide. R. principis crude polysaccharides significantly decreased the levels of TNF-α and interleukin-6 in both serum and blood and bronchoalveolar lavage fluid in lipopolysaccharide-induced acute lung injury mice by intragastric administration (100 mg/kg). A heteropolysaccharide, ZNDHP, was obtained from R. principis crude polysaccharides with successive anticomplementary activity-guided separation. ZNDHP was characterized as a branched neutral polysaccharide with a backbone composed of → 2)-ß-Glcp-(1→, → 2,6)-α-Glcp-(1→, → 6,3)-ß-Galp-(1→, → 2,6)-α-Galp-(1→, → 6,2)-ß-Glcp-(1→, → 4)-α-Glcp-(1→, → 5)-ß-Araf-(1→, → 3,5)-α-Araf-(1→, and → 4,6)-ß-Manp-(1→, and the backbone structure was further confirmed by partial acid hydrolysis. In addition to anticomplementary and antioxidant activities, ZNDHP exhibited potent anti-inflammatory activity by significantly inhibiting the secretion of nitric oxide, TNF-α, interleukin-6, and interleukin-1ß of lipopolysaccharide-treated RAW 264.7 cells. However, all of these activities decreased greatly after partially hydrolyzing, indicating the importance of the multibranched structure for its bioactivity. Therefore, ZNDHP might be an important component of R. principis for treating inflammation.

Arabinogalactan enhances Mycobacterium marinum virulence by suppressing host innate immune responses.

Arabinogalactan (AG) participates in forming the cell wall core of mycobacteria, a structure known as the mAGP complex. Few studies have reported the virulence of inartificial AG or its interaction with the host immune system. Using clustered regularly interspaced short palindromic repeats interference gene editing technology, conditional Mycobacterium marinum mutants were constructed with a low expression of embA or glfT2 (EmbA_KD or GlfT2_KD), which are separately involved in the biosynthesis of AG arabinose and galactose domains. High-performance gel permeation chromatography and high-performance liquid chromatography assays confirmed that the EmbA_KD strain showed a remarkable decrease in AG content with fragmentary arabinose chains, and the GlfT2_KD strain displayed less reduction in content with cut-down galactose chains. Based on transmission and scanning electron microscopy observations, the cell walls of the two mutants were found to be dramatically thickened, and the boundaries of different layers were more distinct. Phenotypes including the over-secretion of extracellular substances and enhanced spreading motility with a concomitant decreased resistance to ethambutol appeared in the EmbA_KD strain. The EmbA_KD and GlfT2_KD strains displayed limited intracellular proliferation after infecting murine J774A.1 macrophages. The disease progression infected with the EmbA_KD or GlfT2_KD strain significantly slowed down in zebrafish/murine tail infection models as well. Through transcriptome profiling, macrophages infected by EmbA_KD/GlfT2_KD strains showed enhanced oxidative metabolism. The cell survival measured using the CCK8 assay of macrophages exposed to the EmbA_KD strain was upregulated and consistent with the pathway enrichment analysis of differentially expressed genes in terms of cell cycle/apoptosis. The overexpression of C/EBPß and the increasing secretion of proinflammatory cytokines were validated in the macrophages infected by the EmbA_KD mutant. In conclusion, the AG of Mycobacterium appears to restrain the host innate immune responses to enhance intracellular proliferation by interfering with oxidative metabolism and causing macrophage death. The arabinose chains of AG influence the Mycobacterium virulence and pathogenicity to a greater extent.

An oligosaccharide-marker approach to quantify specific polysaccharides in herbal formula by LC-qTOF-MS: Danggui Buxue Tang, a case study.

Polysaccharides have broad bioactivities and are major components of water decoction of herb formulae. However, the quality control of polysaccharides remains a challenge. Oligosaccharide-fragment approach has been considered in elucidating chemical structures of polysaccharides, but never been used for quantitation. Using reference chemicals and a real sample Danggui Buxue Tang (DBT) in this study, an oligosaccharide-marker approach was established to quantify specific polysaccharides. Firstly, linear relationships between parent polysaccharides and hydrolysis-produced daughter oligosaccharides were verified using reference polysaccharides. Then in case of DBT, two fluorescence-labeled oligosaccharides with high specificity to individual parent polysaccharides were selected as markers. They were easily isolated and identified. Their potential in quantification of parent polysaccharides were satisfactorily validated in terms of linearity (r≥0.99), repeatability (RSD ≤ 8.4 %), and spike recovery (≥80 %). This method could be a promising approach for quality assessment of polysaccharides in herbal formulae.

Oligosaccharide-marker approach for qualitative and quantitative analysis of specific polysaccharide in herb formula by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry: Dendrobium officinale, a case study.

Qualitative and quantitative analysis of polysaccharides in herb formula remain challenge due to the limited choices of analytical methods concerning the intrinsic characteristics of large molecular mass. Herein, an oligosaccharide-marker approach was newly developed for quality assessment of polysaccharides in herbal materials, using Dendrobium officinale as a case study. This method involved partial acid hydrolysis of D. officinale polysaccharide (DOP) followed by p-aminobenzoic ethyl ester (ABEE) derivatization. Two ABEE-labeled oligosaccharides namely, Te-Man-ABEE and Pen-Man-ABEE, were selected as chemical markers due to their high specificity in herb formula. The linear relationship between the content of these two markers and the content of DOP was then successfully established respectively. The linear relationship was further transformed to that between peak area of chemical markers and DOP content so that chemical markers were not necessary to be isolated for analysis. This linear relationship was systemically validated in terms of precision and accuracy. The results showed that these two oligosaccharide-markers presented a good linear relationship with DOP (R2 ≥ 0.997) in the range of 0.68-16.02 µg. These markers also demonstrated satisfactory precision (RSD < 7.0%), and recovery (91.41%-118.30%) in real sample determination. Additionally, there was no significant difference between the results given by the two chemical markers as the RSD values were not more than 7.0%. While concerning the results given by the oligosaccharide-markers and the previously-published polysaccharide marker, the RSD value was not more than 6.4%. These suggest that the oligosaccharide-marker approach is a simple, quick, and reliable method to qualitatively and quantitatively determine of specific polysaccharide in herb formula.