4.1 Materials

Lobetyolin was isolated from the fruits of C. lancifolius. Briefly, dried fruits were pulverized and subjected to ethanol extraction, followed by fractionation using column chromatography (silica gel and reversed-phase HPLC) to yield purified Lobetyolin. Additionally, high-purity Lobetyolin (≥ 98% as determined by HPLC) was commercially sourced from Shanghai Yingxin Laboratory Equipment Co., Ltd. (Shanghai, China). The chemical structure of both isolated and purchased Lobetyolin was verified through spectroscopic analyses, including 1H and 13C NMR (Fig. S3 in the Supplementary Material) [35]. β-Mercaptoethanol and Tris-base were sourced from Aladdin Biochemical Technology Co., LTD. (Shanghai, China). Riboflavin T (ThT), paraformaldehyde, Triton X-100, and 5-hydroxytryptamine (5-HT) were sourced from McLean Biochemical Technology Co., LTD. (Shanghai, China). The dimethyl sulfoxide (DMSO) was acquired from Solarbio Life Sciences Co., LTD. (Beijing, China).

4.2 C.elegans strain and maintenance conditions

All C. elegans strains (wild-type N2; CL4176, dvIs27 [myo-3p::Aβ1–42::let-851 3′utr) + rol-6 (su1006)]; CL2006, dvIs2 [pCL12 (unc-54/human Aβ1–42 minigene) + ROre-6 (su1006)]; CL2122, dvIs15 [(pPD30.38) unc-54 (vector) + (pCL26) mtl-2::GFP]; CL2355, dvIs50 [pCL45 (snb-1::Aβ1–42: 3′utr (long) + mtl-2::GFP]) were sourced from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota, MN, USA. The uracil-auxotrophic Escherichia coli OP50 strain, used as a food source for the nematodes, was purchased from Shangyuan Bioscience (Fujian, China). Supplementary Table S6 offers a complete overview of the C. elegans strains utilized in this study.

4.3 Toxicity evaluation in C.elegans

Acute toxicity assays were conducted on synchronized L4-stage N2 C. elegans [36]. Lobetyolin and resveratrol (positive control) were diluted in K medium (32 mM KCl, 51 mM NaCl) to final concentrations of 12.5, 25, and 50 μM for Lobetyolin, and 100 μM for resveratrol. In 96-well plates, 200 μL of each test solution was added per well, followed by 30 worms. The control group received K medium containing 0.1% DMSO. The plates were maintained at 20 °C for a duration of 24 h, following which the quantity of deceased nematodes in each group was documented via microscopic observation.

4.4 Body length determination

We followed a previously reported protocol with minor modifications [14]. Briefly, synchronized L4-stage N2 and CL4176 C. elegans were transferred to nematode growth medium (NGM) plates supplemented with control (0.1% DMSO), Lobetyolin (12.5, 25, or 50 μM), or positive control (100 μM resveratrol). N2 worms were cultured at 20 °C for 4 days, whereas CL4176 worms were kept at 15 °C for 6 days. Body lengths were imaged utilizing an inverted fluorescence microscope (DMi8, Leica, Germany). Image analysis was performed using ImageJ 1.54f (National Institutes of Health, USA) to quantify worm body lengths.

4.5 Reproductive evaluation in C.elegans

The synchronized L4-stage wild-type nematodes were relocated to a new NGM medium containing the control (0.1% DMSO), Lobetyolin (12.5, 25, and 50 μM), and positive control (100 μM resveratrol), a single worm being placed on each plate. To distinguish the offspring from the parent worms, the worms were transferred to fresh plates daily until the end of the reproductive period. The plates were kept at 20 °C, and the number of offspring was counted once they reached the L3 stage.

4.6 Life span determination of C.elegans

The synchronized L4 N2/CL4176 worms were transferred to NGM plates containing control (0.1% DMSO), Lobetyolin (12.5, 25, and 50 μM), and positive control (100 μM resveratrol). The N2 and CL4176 worms were respectively cultivated at 20 °C and 15 °C with at least 60 nematodes per plate. NGM plates were replaced every two days during incubation until all worms had perished. Daily observations recorded the number of normally deceased worms. Death was confirmed by the absence of response to gentle stimulation with a platinum wire. Worms that crawled to the plate edge and desiccated were considered abnormal deaths.

4.7 Determination of lipofuscin in C.elegans

Synchronized L4-stage N2 worms were relocated to NGM plates supplemented with control (0.1% DMSO), Lobetyolin (12.5, 25, or 50 μM), or resveratrol (100 μM, positive control) and cultured for 10 days at 20 °C. Worms were collected, anesthetized with 5 mM levamisole, and mounted on glass slides. Individual worms were imaged utilizing a DMi8 inverted fluorescence microscope (Leica, Germany) at 530 nm excitation. Lipofuscin autofluorescence was captured for ≥ 30 worms per group, with fluorescence intensity quantified via ImageJ software.

4.8 Paralysis rate assay in Aβ-transgenic CL4176

The transgenic strain CL4176 expresses human Aβ1–42 upon temperature upshift from 15 °C to 25 °C, inducing an AD-like paralysis phenotype while maintaining normal growth at 15 °C. This model was employed to evaluate the protective effects against paralysis for Lobetyolin. More than 40 synchronized CL4176 eggs per group were transferred to NGM plates supplemented with control (0.1% DMSO), Lobetyolin (12.5, 25, or 50 μM), or resveratrol (100 μM, positive control). The plates were initially incubated at 15 °C for 48 h and then shifted to 25 °C for an additional 24 h to trigger Aβ expression. Subsequently, the non-paralysis rates were assessed every 2 h. Paralysis was determined as the condition where the worms exhibited only head movement and no body response to repeated stimulation with a platinum wire [11].

4.9 Assessment of serotonin hypersensitivity in transgenic C. elegans

The CL2355 strain expresses human Aβ1–42 in neurons, leading to a paralytic response to exogenous serotonin (5-hydroxytryptamine, 5-HT). In contrast, the CL2122 strain, which lacks neuronal Aβ1–42 expression, serves as a control strain that does not respond to serotonin stimulation [37]. Synchronized eggs from the CL2355 and CL2122 strains were relocated to NGM plates supplemented with control (0.1% DMSO), Lobetyolin (12.5, 25, or 50 μM), or resveratrol (100 μM, positive control). The plates were initially incubated at 15 °C for 3.5 days and then shifted to 25 °C for 1.5 days. Subsequently, 30 worms from each group were exposed to a 5 mg/mL solution of 5-HT for 5 min, and the paralysis rates were assessed based on the lack of movement.

4.10 Quantification of Aβ deposits in C. elegans

Aβ deposition in worms was assessed using Thioflavin T (ThT) staining. Synchronized CL2006 eggs were transferred to NGM plates supplemented with control (0.1% DMSO), Lobetyolin (12.5, 25, or 50 μM), or resveratrol (100 μM, positive control) and incubated at 20 °C until day 8 of adulthood. The worms were harvested, washed with M9 buffer, and subsequently fixed in a 4% paraformaldehyde solution at 4 °C for 24 h. After performing three washes with M9 buffer, permeabilization was carried out using an infiltration solution composed of 1% Triton X-100, 5% β-mercaptoethanol, and 125 mM Tris–HCl (pH 7.4) at 37 °C for 24 h. The worms were then washed three times with M9 buffer and stained with 0.125% Thioflavin T (ThT) for 2 min. Background fluorescence was eliminated through repeated washes with 50% ethanol. Finally, the worms were mounted onto slides and imaged using an inverted fluorescence microscope (DMi8, Leica, Germany). Aβ plaques were quantified by counting ThT-reactive deposits in the anterior region (head) of 30 worms per group.

4.11 Quantification of ROS in C. elegans

Intracellular reactive ROS levels were assessed using the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Synchronized N2 and CL4176 eggs were plated on NGM with control (0.1% DMSO), Lobetyolin (12.5, 25, or 50 μM), or resveratrol (100 μM) and incubated at 20 °C (N2) or 15 °C (CL4176). Adult worms (day 8 N2; day 10 CL4176) were placed in 96-well plates with M9 buffer containing 50 μM DCFH-DA, then incubated dark for 2 h at respective temperatures. After M9 washes, worms were anesthetized (5 mM levamisole) on slides and imaged via DMi8 microscope (Leica, Germany) at 470 nm excitation, with 30 worms per group. Fluorescence intensity was quantified with ImageJ.

4.12 Sample preparation for LC/MS metabolomics analysis

Metabolomics samples were divided into G1 (CL4176 with 0.1% DMSO, AD model) and G2 (CL4176 with 50 μM Lobetyolin), with 5 replicates each. Synchronized eggs were seeded onto appropriate NGM plates, cultured at 15 °C for 48 h, and then moved to 25 °C to trigger Aβ expression. Worms were harvested at 26 h post-induction (G2 at 90% non-paralysis time), washed with M9 buffer and distilled water, rapidly frozen in liquid Nitrogen, and subsequently freeze-dried. Dry weights were recorded. Metabolites were extracted using 80% methanol with 10 μM 2-bromo-L-phenylalanine (internal standard) at a 1:200 (mg: μL) ratio. Samples were homogenized on ice, centrifuged at 12,000 rpm for 10 min at 4 °C, and supernatants collected. QC samples were prepared by pooling 10 μL from each. All were stored at − 20 °C before testing.

4.13 LC–MS/MS analysis and data processing

Samples and QC samples were analyzed via LC–MS/MS on a Q Exactive Focus Orbitrap system (Thermo Fisher Scientific, USA) [38]. Briefly, 5 μL samples were separated on a Hypersil Gold™ aQ C18 column (150 × 2.1 mm, 1.9 μm) at 30 °C, eluting with a gradient of MeCN (containing 0.1% AcOH) at 0.3 mL/min. MS scans in positive/negative modes used ddMS2 (m/z 70–1000), with MS1/MS2 resolutions of 70,000/35,000 and stepped collisions (10, 20, 40 V).

All supernatants and QC samples were injected into LC–MS/MS for analysis with analytical parameters set according to previously reported methods [38]. In summary, LC–MS/MS analysis was conducted using a Q Exactive Focus Orbitrap LC–MS/MS system (Thermo Fisher Scientific, Waltham, MA, USA). The 5.0 μL sample was separated on an aQ C18 Polar column of Hypersil Gold™ (1.9 μm, 150 × 2.1 mm) maintained at 30 °C. The mobile phase consisted of H2O (A)-acetonitrile (B), both of which contained 0.1% (v/v) acetic acid. The column was eluted with a gradient (1–100% in 15 min) of Mobile Phase B at a flow rate of 0.3 mL/min. Mass spectra were scanned in positive and negative ion modes with step collisions (10, 20, and 40 V) using the ddMS2 scheme. Full MS and MS/MS scans ranged from 70 to 1000 m/z, with MS1 solution 70000 and MS2 35000.

Data analysis and visualization were performed with reference to a previous study from our laboratory, with minor modifications [39]. The mixOmics package in Bioconductor R software was utilized for PCA and OPLS-DA analyses. Statistical p-values were determined using a t-test in R software. The thresholds of FC > 1.5 2 and p < 0.02 were used to screen differential metabolites. Volcano diagrams were generated using the R software package. Finally, using online tools MetaboAnalyst 5.0 [40] and FELLA package (version 1.14) [41] to determine the differences in metabolite pathways of enrichment and analysis.

4.14 Transcriptomics analyses of transgenic CL4176 C. elegans

Sample preparation and collection methods for transcriptomics were the same as those for metabolomics, as in 4.12. The sample data were analyzed in the BGI system on https://biosys.bgi.com (accessed on May 15, 2025) for genetic screening of differentially expressed genes (DEGs) and enrichment.

4.15 RT-qPCR analysis in transgenic CL4176

The collected samples were processed identically to those in the metabolomics analysis, as detailed in Sect. 4.12, and were divided into two groups: the blank controls and the Lobetyolin treatment group, each with 3 replicates. Primer design was carried out by Shenggong Biotechnology Co., Ltd. (Shanghai, China). Total RNA extraction was performed using the AG RNAex Pro reagent (Accurate Biology, Changsha, China) following the manufacturer's protocol. RT-qPCR was performed on a CFX 96 Touch instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative expression levels were determined using the 2−∆∆Ct and normalized to reference genes.

4.16 Determination of Lobetyolin content in different Chinese medicinal materials

The standard curve was drawn, and the standard Lobetyolin (98%, Shanghai Yingxin Laboratory Equipment Co., Ltd. Shanghai, China) was accurately prepared into 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, 0.05 mg/ml and 0.01 mg/ml standard solutions with methanol, and the detection wavelength was set at 210 nm. Then the chromatographic conditions were screened (equal elution with 47% methanol), and the peak area was measured by Agilent 1100 HPLC (Agilent Technologies Co., Ltd, Santa Clara, CA, USA) as the ordinate, and the standard curve was drawn according to the actual mass concentration of Lobetyolin. After that, the sample solution was prepared and added with 100% methanol at a ratio of 1:5 (g: ml), sonicated for 40 min, and then mixed after cooling. The supernatant was taken and filtered to obtain the sample solution. The peak area was measured and substituted into the standard curve to calculate the content of codonopsis side in Chinese herbal medicine.

4.17 Data analysis

All the data were analyzed with R packages and visualized using ggplot2. One-way ANOVA was performed and followed by Dunnett's multiple comparisons test or log-rank test. Data are presented as mean ± SEM. Asterisks (*) indicate significant differences (ns, not significant; *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).