Anti-aging Activity of Prepared Polygonatum sibiricum Based on Structural Modification of Low-Molecular-Weight Polysaccharides

Different from most current anti-aging studies on polysaccharides that mainly focus on the overall activity of crude polysaccharides or total polysaccharide extracts, this study systematically isolated and purified polysaccharide fractions from steamed Polygonatum sibiricum, obtained homogeneous low-molecular-weight polysaccharide fractions step by step, and thoroughly clarified the molecular mechanisms of its anti-aging effects, including regulating gut microbiota, activating glycolysis pathway, and alleviating Aβ- and Tau-induced neurotoxicity. This study provides new insights into the functional analysis of Polygonatum sibiricum polysaccharides, deepens the understanding of the material basis for the anti-aging activity of steamed Polygonatum sibiricum, and is an important milestone in the research field of Polygonatum sibiricum polysaccharides.

Crude polysaccharides (PSP) were prepared from steamed Polygonatum sibiricum by water extraction and alcohol precipitation: 100 g of steamed Polygonatum sibiricum was extracted with water at 80 °C for 3 times, 2 h each time. The combined filtrate was concentrated, followed by addition of 80% ethanol and precipitation at 4 °C overnight. The precipitate was dissolved in 400 mL of ultrapure water, deproteinized with Sevag reagent (chloroform: n-butanol = 5:1), then purified by adsorption with 10% (w/v) activated carbon. After vacuum filtration, alcohol precipitation was performed again, and freeze-drying was conducted to obtain crude polysaccharides PSP. The yield was 5.41% (w/w), with protein content of 3.2% and sugar content of 95.3%. Deproteinized PSP was stepwise eluted with DE-52 cellulose column (water and 0.03, 0.05, 0.1, 0.3, 0.5 M NaCl solutions). Monitored by phenol-sulfuric acid method, three fractions were obtained: PSP-1 (26.0% of PSP), PSP-2 (34.65%) and PSP-3 (21.3%). PSP-1 and PSP-2 were further purified by Sephadex G-200 gel permeation chromatography column to obtain homogeneous fractions PSP-1-1, PSP-2-1 and PSP-2-2, among which PSP-2-2 was not included in subsequent studies due to low yield. The biological activity of each fraction was evaluated by CL4176 nematode model. The results showed that 100 μg/mL PSP-1 and 150 μg/mL PSP-2 significantly prolonged the paralysis time of nematodes, with PT50 values increased by 42.5% and 40.6% compared with the control group, respectively. Further studies showed that 100 μg/mL PSP-1-1 extended the mean paralysis time by 51.42% compared with the control group, while PSP-2-1 only showed significant activity at a high concentration of 300 μg/mL. Therefore, 100 μg/mL PSP-1-1 was selected for subsequent mechanism research.

Figure 1 Extraction and Isolation/Purification Process of Polysaccharides from Steamed Polygonatum sibiricum.

High-performance gel permeation chromatography (HPGPC) showed that the average molecular weight of PSP-1-1 was 2.7 kDa and the polydispersity index (PDI) was 1.3878, indicating a homogeneous polysaccharide. The total sugar content was 99.2% and no protein was detected by phenol-sulfuric acid method and BCA method. Monosaccharide composition analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization high-performance liquid chromatography (HPLC) showed that PSP-1-1 was mainly composed of mannose, galactose and glucose, with molar percentages of 2.366%, 91.992% and 1.407%, respectively, and galactose was the dominant monosaccharide. Methylation analysis combined with gas chromatography-mass spectrometry (GC-MS) showed that the main linkage type was →4)-Galp-(1→ (68.4%), suggesting a linear 1,4-linked galactan main chain. A small amount of →6)-Galp-(1→ (10.7%), →3)-Galp-(1→ (6.8%) and →4,6)-Galp-(1→ (8.4%) were also detected, indicating branching at O-6 position and minor substitution at O-3 position. Nuclear magnetic resonance (NMR) analysis (¹H, ¹³C, HSQC, HMBC) further confirmed that PSP-1-1 was mainly composed of →4)-β-D-Galp-(1→ linkages with branching at C6 position. No signal was observed in the ¹³C NMR spectrum at δ 170–220 ppm, confirming that PSP-1-1 was a neutral polysaccharide.

Figure 2 Structural Characterization of PSP-1-1.

The lifespan-prolonging and health-improving effects of PSP-1-1 were evaluated using wild-type Caenorhabditis elegans N2 as a model. The results showed that treatment with 100 μg/mL PSP-1-1 increased the median lifespan of nematodes by 29.88% compared with the untreated control group. In terms of healthspan parameters, the body length and movement distance of nematodes in the treatment group on day 5 were significantly increased, while the oviposition rate was not significantly affected, indicating that PSP-1-1 could effectively improve the health status of nematodes while prolonging lifespan without affecting reproductive capacity. Furthermore, an oxidative stress model was constructed with 50 mM paraquat. The results showed that 100 μg/mL PSP-1-1 treatment prolonged the mean survival time of nematodes to 46.44 hours, significantly enhancing their resistance to oxidative stress. Intracellular reactive oxygen species (ROS) level detected by H2DCF-DA fluorescent probe showed that ROS level in nematodes of PSP-1-1 treatment group was reduced by 21.36% compared with the control group, suggesting that PSP-1-1 exerted lifespan-prolonging effect by reducing ROS accumulation.

Figure 3 Evaluation of the Anti-Aging Activity of PSP-1-1 in the Caenorhabditis elegans Model.

In the transgenic CL4176 nematode model expressing human Aβ peptide, 100 μg/mL PSP-1-1 treatment prolonged the mean paralysis time by 51.42% compared with the control group. Western blot results showed that it significantly reduced the protein levels of Aβ oligomers and monomers, indicating that PSP-1-1 could alleviate Aβ-induced neurotoxicity and inhibit Aβ deposition. In the CL2355 transgenic nematode model with pan-neuronal Aβ expression, PSP-1-1 treatment significantly improved the chemotaxis index and ameliorated Aβ-induced chemotactic behavioral defects. Meanwhile, the proportion of nematodes with normal response in 5-hydroxytryptamine sensitivity assay increased from 25.1%±2% in the control group to 47.1%±2%. In addition, in PHX3692 transgenic nematodes with mCherry-labeled D-type motor neurons, PSP-1-1 treatment significantly increased the number of D-type motor neurons and reduced the proportion of neuronal damage, suggesting its protective effect on neuronal integrity. In the VH254 transgenic nematode model (exhibiting age-dependent motor dysfunction mediated by Tau hyperphosphorylation), 100 μg/mL PSP-1-1 treatment significantly increased the body bend frequency of nematodes and effectively ameliorated motor dysfunction caused by Tau pathological changes.

Figure 4 Assessment of the Neuroprotective Effects of PSP-1-1.

16S rRNA gene sequencing was used to compare the regulatory effects of PSP-1-1 treatment group, inulin (positive control) group and blank control group on human gut microbiota. The results showed that about 1670 operational taxonomic units (OTUs) were obtained in the PSP-1-1 group, and its microbial community composition was significantly changed compared with the control group. Alpha diversity analysis (Chao index) showed that the species diversity of gut microbiota in the PSP-1-1 group was significantly increased. At the phylum level, PSP-1-1 treatment increased the relative abundance of Firmicutes and decreased the relative abundance of Bacteroidetes and Proteobacteria; at the genus level, the abundance of Lactobacillus was significantly increased. In addition, PSP-1-1 treatment also significantly increased the contents of short-chain fatty acids (SCFAs), including acetic acid, propionic acid and butyric acid.

Figure 5 Regulatory Effects of PSP-1-1 on Gut Microbiota and Short-Chain Fatty Acids.

KEGG pathway enrichment analysis showed that PSP-1-1 could significantly activate the "carbohydrate metabolism" pathway in the secondary metabolic pathway. To further verify the changes of this pathway, real-time quantitative PCR was used to detect the mRNA expression levels of key glycolysis-related genes in Caenorhabditis elegans. The results showed that PSP-1-1 treatment significantly up-regulated the transcription levels of pfk-1.1, gpi-1, aldo-1, aldo-2 and pyl-1 genes. Among them, pfk-1.1 encodes phosphofructokinase, the rate-limiting enzyme of glycolysis pathway, and its increased expression helps to enhance glycolysis flux and promote intracellular energy metabolism and ATP production. In conclusion, PSP-1-1 may exert the biological effect of delaying aging by activating glycolysis pathway and improving the body's energy metabolism status.

Figure 6 Mechanistic Study of PSP-1-1-Mediated Anti-Aging Effects via the Glycolytic Pathway.

Summary

This study successfully isolated and purified a low-molecular-weight polysaccharide named PSP-1-1 from steamed Polygonatum sibiricum, which is a homogeneous polysaccharide with 1,4-β-D-galactan as the main chain. In vivo experiments using Caenorhabditis elegans as a model, PSP-1-1 showed significant anti-aging activity, specifically manifested as prolonging nematode lifespan, improving healthspan, enhancing antioxidant capacity and stress tolerance. In addition, PSP-1-1 can effectively alleviate neurotoxicity induced by pathological changes of Aβ and Tau proteins, protect neuronal structural integrity, and improve motor dysfunction caused by neurodegeneration. Mechanism studies showed that the anti-aging effect of PSP-1-1 may be related to regulating gut microbial composition, including increasing the relative abundance of beneficial bacteria (such as Lactobacillus) and promoting the production of short-chain fatty acids (SCFAs); meanwhile, PSP-1-1 can also activate glycolysis metabolic pathway and enhance energy metabolism level. In summary, PSP-1-1 shows potential as a natural anti-aging functional factor, has broad application prospects in the development of anti-aging drugs, functional foods and nutritional health products, and provides new research ideas and experimental basis for promoting healthy aging and preventing age-related diseases.

Reference: Zhang C, Lin H, Wei C, et al. Anti-aging effects of low-molecular-weight polysaccharide PSP-1-1 from steamed Polygonatum sibiricum via gut microbiota modulation[J]. Carbohydr Polym, 2026, 381: 125225.