Selaginella plant materials were collected from 13 provinces in China and cultivated at the understory resource nursery at Hubei University of Chinese Medicine. Details of the collection sites were listed in Supplementary Table S1 and Supplementary Figure S1, and morphological characteristics of select Selaginella specimens were shown in Supplementary Figure S2. Wild-type C. elegans (N2 strain), DAF-16 mutant worms (CF1038), DAF-16GFP (TJ356) and Escherichia coli OP50 were generously provided by Dr. Pan Li from South China Agricultural University.

For molecular experiments, the Total RNA Kit (RC112-01), and HiScript III first Strand cDNA Synthesis Kit (R312-01) were obtained from Vazyme Biotech Co., Ltd. (Nanjing, China). Astaxanthin (catalog number: SA8730), and amentoflavone (catalog number: 1,617-53-4) were sourced from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

The aerial parts of Selaginella were harvested and dried at 60 °C until a constant weight was obtained. The dried plant material was then finely ground and passed through a 65-mesh sieve. For extraction, 100 mg of the powdered sample was soaked in 10 mL of methanol (HPLC Grade, ≥99.9%, FTSCI Hubei) for 40 min. This was followed by ultrasonic extraction using an SB-4200DT ultrasonic cleaner (SCIENTZ, China) at 55 °C for 30 min. The mixture was then filtered through a 0.45 µm membrane (XingYa, Shanghai) to obtain crude extracts, designated as MEs, at a final concentration of 10 mg mL^-1. This ME solution was used as the stock solution and further diluted to 0.5, 1.0, and 2.0 mg mL^-1 for subsequent experiments.

Twenty milligrams of amentoflavone monomer were dissolved in methanol and diluted to a final concentration of 1 mg mL^-1 to prepare a stock solution. This stock solution was then further diluted to concentrations of 25, 50, and 100 μg mL^-1 for use in subsequent experiments. Astaxanthin was prepared separately by diluting in chloroform to a final concentration of 0.64 µM. All solutions were filtered using a 0.45 µm organic filter membrane, aliquoted into 500 µL portions, and stored at −80 °C until further use.

The C. elegans strains were cultured at 20 °C on solid Nematode Growth Medium (NGM) agar plates seeded with Escherichia coli OP50.

For the heat stress resistance assay, 60-69 worms were divided into each experimental group, with each group distributed across three NGM plates (20-23 worms per plate). The plates were supplemented with either 0.5, 1.0 and 2.0 mg mL^-1 of Selaginella methanol extracts (SME) or 50 μg mL^-1 of amentoflavone monomer. The worms were incubated at 37 °C, and the number of survivors was recorded at 1-hour intervals until all the worms perished.

Similarly, in the ultraviolet (UV) stress resistance assay, 60-69 worms were distributed into groups on three NGM plates (20-23 worms per plate), supplemented with either 0.5, 1.0 and 2.0 mg mL^-1 of SME or 50 μg mL^-1 of the monomer. The worms were then exposed to UV irradiation (8 W) for six additional days. Survival rates were recorded at 12-hour intervals until all the worms perished.

For lifespan analysis of wild-type C. elegans (N2 strain) or daf-16 mutant worms (CF1038), the assay were performed following previously described methods (Wang et al., 2024). Worms were synchronized and lysed, then cultured on NGM plates at 20 °C until reaching the L4 larval stage. The L4 larvae were exposed to SME or the monomer to evaluate lifespan. Worms treated with 100% methanol served as the negative control. To prevent progeny development, 5-fluorodeoxyuridine (10 μg mL^-1) was administered from day 0 to day 5. The number of surviving worms was recorded daily, and the worms were transferred to fresh plates every 24 h. Survival curves were analyzed using GraphPad Prism 9 software.

Synchronized L4 C. elegans (N2 strain) were transferred to NGM plates containing the SMEs (0.5, 1.0, 2.0 mg mL^-1) or monomer amentoflavone (50 μg mL^-1). Worms were transferred to fresh NGM plates daily throughout the 5-day treatment period. Afterward, approximately 200 worms were harvested for RNA sequencing analysis.

Total RNA extraction followed the protocol of the R6834 Total RNA Kit I. Libraries were then prepared and sequenced using the DNBSEQ high-throughput sequencing platform (BENAGEN, China). The trimmed sequencing reads were aligned to the C. elegans reference genome (Yoshimura et al., 2019) using STAR software (version 2.7.9a). For transcript assembly, StringTie (version 2.1.4; default parameters) was employed, and the assembled transcripts were merged using StringTie’s “merge” function. The merged transcripts were compared with known genome annotations using gffcompare (version 0.12.1; parameters: R-C-K) to identify novel transcripts and genes, thereby complementing the existing annotations. Transcript abundance and gene quantification were calculated using RSEM software.

Transcript levels were quantified by calculating Fragments Per Kilobase of transcript per Million mapped reads (FPKM), with an FPKM value of 1 as the threshold for transcript expression. Differentially expressed transcripts (DGTs) were identified based on the criteria |logFC| > 1 and p < 0.05. Data visualization and functional enrichment analyses, including volcano plots, heatmaps, GO term enrichment, KEGG pathway analysis, and Gene Set Enrichment Analysis (GSEA), were performed using the clusterProfiler package.

To explore the interactions among the significant transcripts (p < 0.05), a PPI network was constructed. The PPI network was built using the Search Tool for the Retrieval of Interacting Genes (STRING) database (https://www.string-db.org/) and visualized using Cytoscape software.

The HPLC analysis was carried out using an Agilent 1,260 Infinity II LC system (Agilent Technologies Inc.) equipped with an Agilent 5 TC-C18 (2) column (250 × 4.6 mm, 5 μm). The chromatographic conditions were as follows: solution A: 0.1% phosphoric acid water, solution B: 100% methanol (high performance liquid phase grade); Column temperature: 30 °C; Detection wavelength: 330 nm; Injection volume: 10 μL; Flow rate: 1 mL min^-1; Gradient conditions: 0.0 min, 40% B; 14.0 min, 50% B; 19.0 min, 60% B; 24.0 min, 70% B; 29.0 min, 80% B; 49.0 min, 90% B; 51.0 min, 40% B; 53.0 min, 40% B.

Nematodes were maintained under the same culture conditions as described in the lifespan assay. The pharyngeal pumping rate was assessed following a previously reported method (Ding et al., 2024), with slight modifications. Briefly, on days 3 and 7 of cultivation, relaxation and contraction cycles of the pharyngeal pump were observed and counted over a 30-second interval under a microscope. In addition, body bending frequency was evaluated on days 3, 7, and 12 of culture, during which body bends were recorded within a 30-second observation window (Yu et al., 2021).

For the fertility assay, synchronized nematodes were individually transferred to NGM plates—either blank control or amentoflavone (50 μg mL^-1) treatment groups—with three replicates per group. Of note, 5-FU was not included in the NGM plates during this experiment. Every 24 h, the nematodes were transferred to fresh corresponding NGM plates. The number of eggs was recorded once the offspring had developed to the L2 or L3 larval stage, and counting continued until the end of the egg-laying period.

RNA was extracted from three biological replicates, each consisting of approximately 200 worms. Total RNA was isolated using Trizol reagent, and cDNA was synthesized with SuperScript^® II Reverse Transcriptase. RT-qPCR was performed using TB Green^® Premix Ex Taq™ II (Tli RNaseH Plus) on a StepOnePlus™ Real-Time PCR System, in accordance with the manufacturer’s instructions. The housekeeping gene pmp-3 (GenBank accession number NM_001269678.3) was used as an internal control. Primer sequences are provided in Supplementary Table S2.

Intracellular ROS levels were detected according to the method described by Li et al. (2022). N2 wild-type worms were treated with methanol or amentoflavone for 5 days. Subsequently, the worms were collected into 1.5 mL centrifuge tubes using M9 buffer and washed three times. The nematodes were then incubated with 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA; 1 μM, Sigma) at 20 °C for 4 h. After incubation, the worms were washed with M9 buffer, anesthetized with 5 mM levamisole, transferred onto glass slides, and visualized under a fluorescence microscope (Olympus IX73, Tokyo, Japan) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Fluorescence intensity was quantified using ImageJ software (NIH, Bethesda, MD). Each experiment was performed in triplicate with 30 worms per treatment group.

The subcellular localization of DAF-16 was examined as previously reported (Lin et al., 2019). The transgenic strain TJ356 was employed to monitor DAF-16GFP localization. After treatment, worms were washed with M9 buffer and anesthetized using 5 mM levamisole. Images were acquired with a fluorescence microscope and analyzed using ImageJ. DAF-16GFP localization patterns were classified into three categories: cytosolic, intermediate, and nuclear. Assays were conducted in triplicate, with 30 worms analyzed per treatment.

All data were analyzed using GraphPad Prism 9.0. The Log-rank (Mantel-Cox) test was employed for survival analysis, while comparisons between two groups were performed using a two-sided Student’s t-test. For multiple comparisons, two-way ANOVA with Bonferroni’s correction was applied. Results are presented as the mean ± standard deviation (SD). Statistical significance was set at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001.

Selaginella has been widely utilized in traditional medicine and cosmetics due to its bioactive properties, which include promoting blood circulation, regulating menstruation, and enhancing skin whitening (Bailly, 2021; Lee et al., 2022). However, its potential effect on lifespan extension remains unexplored. To address this, we collected 23 Selaginella samples from 14 different provinces across China (Figure 1A; Supplementary Figures S1–S2; Supplementary Table S1), designated as S1 to S23. HPLC analysis revealed substantial variation in the concentration of amentoflavone—a key bioactive biflavonoid and an established chemical marker in Selaginella (Zhang et al., 2022)—across species and geographic origins (Figure 1B; Supplementary Table S3). Based on amentoflavone content, the samples were classified into three groups: high (≥23.92 μg g^-1), medium (11.15–23.49 μg g^-1), and low (≤10.39 μg g^-1) (Figure 1B).

For subsequent lifespan analysis, two representative samples with contrasting amentoflavone levels were selected: S4, which had the second-highest content (33.07 μg g^-1) from the high group, and S16, which had the lowest (4.24 μg g^-1) from the low group. This comparative approach, based on divergent phytochemical profiles, allows for a more precise assessment of the potential role of Selaginella extract and its constituent amentoflavone in modulating lonevity.

As lifespan is a primary biomarker of aging, we evaluated the lifespan extension potential of two distinct Selaginella extracts S4 and S16—in the C. elegans model using a concentration-gradient experiment (0.5, 1.0, and 2.0 mg mL^-1). By day 21, lifespan curves for S4-treated worms exhibited significant rightward shifts compared to the methanol-treated control, corresponding to average lifespan extensions of 8.56%–17.19% (Figure 2A; Table 1). Similarly, by day 27, S16-treated groups showed rightward shifts relative to the control; however, these were less pronounced than those observed with S4, suggesting potential concentration-dependent toxicity in this specific geographical variant (Figure 2B; Table 1). These results demonstrate that Selaginella extracts significantly extend lifespan in wild-type C. elegans, with efficacy varying according to geographic origin and associated phytochemical composition.

To further assess the survival benefits of S4 and S16, we examined their effects on C. elegans under UV stress. S4 treatment resulted in significant rightward shifts in survival curves, increasing lifespan by 1.47%–10.81% under UV exposure (Figure 2C; Table 1). In contrast, low and medium concentrations of S16 caused leftward shifts in the survival curve. The high-concentration S16 group showed no difference from the control under UV stress (Figure 2D; Table 1). These findings indicate that S4 not only extends lifespan under normal conditions but also enhances resilience to UV stress, supporting its lifespan extension potential. Conversely, S16 failed to improve lifespan under UV stress.

To investigate the lifespan extension mechanisms of Selaginella derivatives, we performed RNA-seq analysis on C. elegans treated with S4 or S16. S4 treatment induced 2,439 differentially expressed transcripts (DETs), significantly enriched in MAPK, FOXO, mTOR, and longevity-regulating pathways (Figure 2E; Supplementary Tables S3-S5). S16 treatment altered 1,203 DETs, predominantly enriched in MAPK, Wnt, ErbB, and longevity-regulating pathways (Figure 2F; Supplementary Tables S6-S8). Although S4 induced approximately twofold more DETs than S16, both treatments shared enrichment in MAPK and longevity-regulating pathways, suggesting a common lifespan extension mechanism. This result indicates that Selaginella derivatives extend lifespan through coordinated modulation of evolutionarily conserved longevity pathways and compound-specific metabolic interventions.

Protein interaction network analysis identified five hub genes per treatment, with daf-16, egl-8, and C56A3.8 common to both groups (Figures 2G,H). Functional studies confirmed their critical roles: DAF-16 mediates insulin/IGF-1 signaling in lifespan regulation; egl-8 (typically associated with nicotine dependence studies) encodes a protein that activates 1-phosphatidylinositol 4-kinase and participates in phosphatidylinositol phosphate biosynthesis. These findings suggest both samples converge on FOXO signaling activation (Figures 2I,J).

To identify potential bioactive components, we conducted network pharmacology and molecular docking analyses (Supplementary Figure S3-S6; Supplementary Table S9-S12). The results revealed ten key candidate compounds: andromedotoxin, asebotoxin, beta-caryophyllene, isocembrol, hinokiflavone, isocryptomerin, amentoflavone, selaginellin, hinokinin, and tremetone. Combined with HPLC analysis identifying the amentoflavone as primary active substance in Selaginella, we selected amentoflavone for further mechanistic study (Figure 1A).

In pre-experimental tests, amentoflavone was evaluated at concentrations of 25, 50, and 100 μg mL^-1. The 50 μg mL^-1 concentration demonstrated the most pronounced lifespan extension without any adverse effects, such as alterations in feeding behavior, and was therefore chosen for further mechanistic investigation. Lifespan analysis of C. elegans treated with 50 μg mL^-1 amentoflavone revealed a significant extension of 63.81% (Figure 3B; Table 1). This represents a 3.7-fold greater increase compared to the group treated with S4 SME, identifying amentoflavone as a key contributor to the lifespan-extending properties of Selaginella.

Further assessment under UV, thermal and oxidative stress conditions showed that amentoflavone induced rightward survival curve shifts (Wang et al., 2020; Hou et al., 2023). Lifespan enhancements ranged from 1.47% under UV stress, 18.29% under thermal stress and 19.75% under oxidative stress (Figures 3C–E; Tables 2-4). Notably, the improvement in both thermal and oxidative stress resistances were significantly greater than under UV stress, suggesting this amentoflavone may primarily extend lifespan by enhancing resistance to thermal and oxidative damage.

Collectively, these findings demonstrate that amentoflavone significantly extend C. elegans lifespan and improve thermal and oxidative stress resistances, confirming its role as a key active compound in Selaginella for lifespan extension.

To further explore the mechanisms by which Selaginella polyphenols extend lifespan, we performed RNA sequencing to analyze differentially expressed transcripts (DETs) in C. elegans treated with amentoflavone. We identified 693 DETs (372 upregulated, 267 downregulated) in amentoflavone-treated worms (Supplementary Table S13). Volcano plots and heatmaps of these DETs (FPKM >1, p < 0.05) are presented in Figures 4A,B.

Subsequent Gene Ontology (GO) enrichment analysis categorized these DETs into 3,214 terms, encompassing 2,161 biological processes (BPs), 402 cellular components (CCs), and 651 molecular functions (MFs) (Supplementary Table S14). The top 10 enriched terms for each category are shown in Supplementary Figure S9. The DETs were predominantly associated with BPs including regulation of biological process, cellular process, and response to stimulus. Key CCs included supramolecular complex and cell junction, while primary MFs involved metal ion binding and protein binding (Supplementary Figure S9; Supplementary Table S15).

KEGG pathway analysis revealed significant enrichment (p < 0.05) of these DETs in 8 pathways, among which 5 signaling pathways—MAPK, FOXO, Phosphatidylinositol, mTOR, and ECM-receptor interaction—were relevant to our study objectives (Figure 4C; Supplementary Table S16,S17). Gene set enrichment analysis (GSEA) indicated upregulation of all these signaling pathways (Supplementary Table S18).

To identify key molecular events, we conducted protein-protein interaction (PPI) analysis on DETs related to aging and longevity. Five central hub genes were identified: daf-16, daf-18, daf-15, egl-8, and lin-45 (Figure 4D; Supplementary Table S10). DAF-16, a key transcription factor downstream of insulin/IGF-1 signaling, regulates lifespan (Murphy et al., 2003). DAF-15 deficiency-induced longevity requires intestinal DAF-16/FOXO activity (Zang et al., 2024), while DAF-18 promotes DAF-16 nuclear translocation by inhibiting PIP3 and the PI3K-Akt pathway and is linked to antioxidant activity (Berman and Kenyon, 2006). Furthermore, other hub genes (egl-8, lin-45) are functionally connected to the FOXO pathway: EGL-8 acts upstream of DAF-16 to regulate lifespan in a DAF-16-dependent manner (Mack et al., 2022); LIN-45 (ERK signaling) influences DAF-2/DAF-16 insulin-like signaling via SKN-1 (Okuyama et al., 2010). These findings collectively suggest that amentoflavone extends lifespan by activating the DAF-16/FOXO transcription factor and upregulating the FOXO signaling pathway (Figures 4A–D).

To exclude potential confounding effects of dietary restriction on the IIS signaling pathway in C. elegans, we measured the pharyngeal pumping rate of wild-type worms following amentoflavone treatment. As shown in Figure 4E, a slight increase in pharyngeal pumping frequency was observed in amentoflavone-treated worms compared to the control group, indicating that amentoflavone does not reduce feeding behavior. Moreover, we also assessed the healthspan of C. elegans following amentoflavone treatment, specifically focusing on body bending ability and reproductive capacity. However, the results revealed no significant differences in either body bending or fertility between the amentoflavone-treated group and the blank control group (Supplementary Figure S8).

To further validate the pivotal role of DAF-16, we treated daf-16 knockout C. elegans mutants with amentoflavone (50μg mL-1). No significant alterations in survival curves were observed compared to wild-type controls (Figure 4F), confirming that DAF-16 is essential for the anti-aging effects of this compound. Since nuclear translocation is necessary for DAF-16 function, we used the DAF-16GFP (TJ356) strain to examine whether amentoflavone influences DAF-16 subcellular localization. As illustrated in Figures 5A,B, amentoflavone treatment significantly enhanced nuclear accumulation of DAF-16 by 48.85% compared to the control, supporting the involvement of this transcription factor in lifespan extension.

We next assessed intracellular ROS levels in C. elegans, as reduced ROS is indicative of enhanced antioxidant capacity. Amentoflavone administration resulted in a 50.22% decrease in ROS content compared to controls (Figures 5C,D).

Given the well-established association between FOXO signaling and antioxidant defense (Kim et al., 2014), we investigated DAF-16-interacting genes within protein–protein interaction (PPI) networks (Supplementary Figure S10). Our analysis revealed that DAF-16 interacts with three antioxidant-related genes: mig-1, tax-6, and pck-2 (Supplementary Table S19). Expression analysis indicated that nearly all of these genes (with the exception of mig-1) were significantly upregulated (p < 0.05) following amentoflavone treatment.

To further assess the impact on antioxidant gene expression downstream of FOXO, we performed RT-qPCR on key genes, including Sod-3, Gst-3, Gst-4, Gsto-1, Hsp-16.48, and Hsp-12.6 (Figure 5E). While Sod-3 expression was only mildly increased, members of the Hsp and Gst families showed significant upregulation compared to the control group. These results suggest that amentoflavone enhances antioxidant defenses primarily through the induction of these gene families.

In conclusion, our findings indicate that amentoflavone extends lifespan and improves antioxidant capacity in C. elegans mainly by activating DAF-16/FOXO and upregulating its downstream antioxidant pathway. DAF-16/FOXO-mediated gene regulation appears crucial for the observed longevity phenotype (Figure 5F).