Structural Characterization and Antioxidant Activity of Polysaccharides from Athyrium multidentatum (Doll.) Ching in d-Galactose-Induced Aging Mice via PI3K/AKT Pathway.

The purpose of this study was to characterize the polysaccharides from Athyrium multidentatum (Doll.) Ching (AMC) rhizome and explore the protective mechanism against d-galactose-induced oxidative stress in aging mice. METHODS: A series of experiments, including molecular weight, monosaccharide composition, Fourier transform infrared (FT-IR) spectroscopy, and 1H nuclear magnetic resonance (1H NMR) spectroscopy were carried out to characterize AMC polysaccharides. The mechanism was investigated exploring d-galactose-induced aging mouse model. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and western blotting assays were performed to assess the gene and protein expression in liver. KEY FINDINGS: Our results showed that AMC polysaccharides were mainly composed of mannose (Man), rhamnose (Rha), glucuronic acid (Glc A), glucose (Glc), galactose (Gal), arabinose (Ara), and fucose (Fuc) in a molar ratio of 0.077:0.088:0.09:1:0.375:0.354:0.04 with a molecular weight of 33203 Da (Mw). AMC polysaccharides strikingly reversed d-galactose-induced changes in mice, including upregulated phosphatidylinositol 3-kinase (PI3K), Akt, nuclear factor-erythroid 2-related factor 2 (Nrf2), forkhead box O3a (FOXO3a), and hemeoxygenase-1 (HO-1) mRNA expression, raised Bcl-2/Bax ratio, downregulated caspase-3 mRNA expression, enhanced Akt, phosphorylation of Akt (p-Akt), Nrf2 and HO-1 protein expression, decreased caspase-3, and Bax protein expression. CONCLUSION: AMC polysaccharides attenuated d-galactose-induced oxidative stress and cell apoptosis by activating the PI3K/AKT pathway, which might in part contributed to their anti-aging activity.

Cytoproliferative and Cytoprotective Effects of Striatisporolide A Isolated from Rhizomes of Athyrium multidentatum (Doell.) Ching on Human Umbilical Vein Endothelial Cells.

OBJECTIVES: The aim of this study was to investigate the proliferative and protective effects of striatisporolide A (SA) obtained from the rhizomes of Athyrium multidentatum (Doell.) Ching on human umbilical vein endothelial cells (HUVECs). METHODS: Cell viability was measured by the MTT method. Cell apoptosis was determined by flow cytometry. Intracellular ROS was measured by the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. RESULTS: The viability rate in cells treated with 100 µM SA alone was increased to 128.72% ± 0.19% and showed a significant difference compared with the control group (p < 0.05). Meanwhile, SA augmented the cell viabilities in H2O2-treated HUVECs, and the cell viability was enhanced to 56.94% ± 0.13% (p < 0.01) when pre-incubated with 50 µM SA. The cell apoptosis rates were reduced to 2.17% ± 0.20% (p < 0.05) and 3.1% ± 0.34% (p < 0.01), respectively, after treatment with SA alone or SA/H2O2. SA inhibited the overproduction of reactive oxygen species (ROS) in HUVECs induced by H2O2 and the fluorescent intensity was abated to 9.47 ± 0.61 after pre-incubated with 100 µM SA. CONCLUSIONS: The biological activities of SA were explored for the first time. Our results stated that SA exhibited significant cytoproliferative and minor cytoprotective effects on HUVECs. We presume that the mechanisms of the proliferation and protection actions of SA involve interference with the generation of ROS and the cell apoptosis. These findings provide a new perspective on the biological potential of butenolides.

The antihyperlipidemic mechanism of high sulfate content ulvan in rats.

Numerous studies have suggested that hyperlipidemia is closely linked to cardiovascular disease. The aim of this study was to investigate the possible antihyperlipidemia mechanism of HU (high sulfate content of ulvan) in high-cholesterol fed rats. Wistar rats were made hyperlipidemic by feeding with a high-cholesterol diet. HU was administered to these hyperlipidemia rats for 30 days. Lipid levels and the mRNA expressions of FXR, LXR and PPARγ in liver were measured after 30 days of treatment. In the HU-treated groups, the middle dosage group of male rats (total cholesterol (TC): p < 0.01) and the low-dosage group of female rats (TC, LDL-C: p < 0.01) showed stronger activity with respect to antihyperlipidemia. Moreover, some HU groups could upregulate the mRNA expression of FXR and PPARγ and downregulate the expression of LXR. For the male rats, compared with the hyperlipidemia group, the middle dosage HU had the most pronounced effect on increasing the mRNA levels of FXR (p < 0.01); low- and high-dosage HU showed a significant inhibition of the mRNA levels of LXR (p < 0.01). All HU female groups could upregulate the mRNA expression of PPARγ in a concentration-dependent manner. In summary, HU could improve lipid profiles through upregulation of FXR and PPARγ and downregulation of LXR.

Protective effect of phosphorylated Athyrium multidentatum (Doll.) Ching polysaccharide on vascular endothelial cells in vitro and in vivo.

The purpose of this study was to prepare phosphorylated Athyrium multidentatum (Doll.) Ching polysaccharide (PPS) and investigate its protective effect on vascular endothelial cells (VECs) in vitro and in vivo and the underlying mechanisms. Sodium tripolyphosphate (STPP) and sodium trimetaphosphate (STMP) were used as phosphorylation reagents and PPS was characterized by Fourier transform infrared (FT-IR), 13 C nuclear magnetic resonance (13 C NMR) and 31 P nuclear magnetic resonance (31 P NMR) spectra. Chemical analysis demonstrated that PPS was composed of mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose with a molar ratio of 11.36:0.42:4.03:1.12:1.81:0.26:33.25:24.12:6.85:14.46:2.32 and a molecular weight of 28,837 Da. Results from in vitro and in vivo assays revealed that PPS protected human umbilical vein endothelial cells (HUVECs) against H2 O2 -induced oxidative injury and attenuated D-galactose-induced VECs damage in mice. RNA sequencing (RNA-seq) analysis identified 18 differentially expressed genes (DEGs) between D-galactose-treated and PPS-pretreated mice abdominal aorta. A deep analysis of these DEGs disclosed that PPS regulated the expression of genes involved in the functions of vascular endothelium repairment, cell growth and proliferation, cell survival and apoptosis, inflammation, angiogenesis and antioxidant, indicating that these biological processes might play crucial roles in the protective actions of PPS on VECs.

Structural characteristics and immune-enhancing activity of fractionated polysaccharides from Athyrium Multidentatum (Doll.) Ching.

Polysaccharides coded as CP were extracted from Athyrium Multidentatum (Doll.) Ching and then fractionated into five fractions (FP-1, FP-2, FP-3, FP-4 and FP-5). A purified polysaccharide designated as FP-3-4 was prepared from FP-3 by Sephadex G-100 column chromatography. Chemical analysis disclosed that CP and these fractions were heteropolysaccharides and mainly composed of glucose, galactose, arabinose, mannose, rhamnose, xylose, fucose, ribose and uronic acid with different molar ratios. They presented different images of SEM. FP-3-4 was highly branched polymers with sixteen types of linkages. The in vitro immunomodulatory results stated that CP and these fractions could promote macrophage proliferation, enhance macrophage phagocytosis and increase the production of NO, TNF-α, IFN-γ, IL-1ß, IL-6, IL-10 and IL-2, indicating remarkable immune enhancement activities. RNA sequencing analysis revealed that CP and FP-3 induced macrophage activation mainly through MAPK and alternative NF-κΒ signaling pathways via CD14/TLR4 and Dectin-2 receptors, which were verified by RT-qPCR and western blot.

Chemical characteristics and cytoprotective activities of polysaccharide fractions from Athyrium Multidentatum (Doll.) Ching.

Five polysaccharide fractions (PS-1, PS-2, PS-3, PS-4 and PS-5) were successfully isolated from Athyrium Multidentatum (Doll.) Ching by anion-exchange column chromatography. Their in vitro cytoprotective activities and the underlying mechanisms were explored in this paper. Chemical analysis suggested that the five polysaccharide fractions were heteropolysaccharides with different molecular weights and monosaccharide compositions. Treatment with these polysaccharide fractions could increase cell viabilities, superoxide dismutase/catalase activities, nitric oxide contents, mitochondrial membrane potential levels and Bcl-2/Bax ratios, and reduce cell apoptosis, intracellular reactive oxygen species production and malondialdehyde contents in H2O2-damaged cells. Moreover, these polysaccharide fractions enhanced the mRNA expression levels of PI3K, Akt, FOXO3a, Nrf2 and HO-1 and PS-4 exhibited the most powerful effects on the mRNA expression of these genes. Current findings suggested that the polysaccharide fractions decreased H2O2-induced apoptosis of HUVECs. The activation of PI3K/Akt/FOXO3a and Nrf2/HO-1 signaling pathways might be involved in the protective mechanisms of the active fractions. The polysaccharides might be one of the key bioactive ingredients of Athyrium Multidentatum (Doll.) Ching for the treatment of oxidative damage.

Striatisporolide A, a butenolide metabolite from Athyrium multidentatum (Doll.) Ching, as a potential antibacterial agent.

The present study aimed to investigate the antibacterial activity of striatisporolide A (SA) against Escherichia coli (E. coli) and the underlying mechanism. Antibacterial activity was evaluated according to the inhibitory rate and zone of inhibition. The antibacterial mechanism was investigated by analyzing alkaline phosphatase (AKP) activity and ATP leakage, protein expression, cell morphology and intracellular alterations in E. coli. The results demonstrated that SA exerted bacteriostatic effects on E. coli in vitro. AKP activity and ATP leakage analysis revealed that SA damaged the cell wall and cell membrane of E. coli. SDS­PAGE analysis indicated that SA notably altered the level of 10 and 35 kDa proteins. Scanning electron microscopy and transmission electron microscopy analyses revealed marked alterations in the morphology and ultrastructure of E. coli following treatment with SA. The mechanism underlying the antimicrobial effects of SA against E. coli may be attributed to its actions of disrupting the cell membrane and cell wall and regulation of protein level. The findings of the present study provide novel insight into the antimicrobial activity of SA as a potential natural antibacterial agent.

Antioxidant properties of different molecular weight polysaccharides from Athyrium multidentatum (Doll.) Ching.

Different molecular weight polysaccharides were prepared by degradation of polysaccharides extracted from Athyrium multidentatum (Doll.) Ching rhizome (CPA) with hydrogen peroxide and ascorbic acid. Four low molecular polysaccharides derivatives (CPA-1, CPA-2, CPA-3 and CPA-4) were successfully obtained and had their antioxidant activities investigated employing various established in vitro systems. All CPA derivatives showed pronounced antioxidant activity, and had stronger antioxidant ability than CPA in certain tests. CPA-1 exhibited the strongest scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical among all samples, and the IC50 value was 25 µg/mL. CPA-2 possessed the highest scavenging ability against superoxide radical at 200 µg/mL. The scavenging activity of CPA-4 on hydroxyl radical was higher than CPA from 120 to 200 µg/mL. The mechanism on influence the antioxidant activity of CPA and its degraded derivatives was indicated.

Antioxidant activity of polysaccharide fractions extracted from Athyrium multidentatum (Doll.) Ching.

Crude polysaccharides were extracted from Athyrium multidentatum (Doll.) Ching (AMC) rhizome and fractionated by DEAE-Cellulose 52 ion-exchange column chromatography. Two polysaccharide fractions (F1 and F2) were obtained and had their antioxidant activities investigated employing various established in vitro systems. All fractions possessed considerable antioxidant activity. Chemical analysis suggested that F1 and F2 were neutral heteropolysaccharide in which glucose was the major component. Available data suggested that the molecular weight and sulfate content played very important roles on antioxidant activity. Our results indicated that the polysaccharides may contribute to the medicinal functions of AMC.