Extract of Sapindus saponaria L., a Native Amazonian Plant, Impacts Germ Cell Development and Modulates Longevity
Ana Carolina Anchieta Adriano, Péterson Alves Santos, Átila Bezerra de Mira, Juliana Souza Terada Nascimento, Patrícia Pereira, Sandro de Vargas Schons
TL;DR
This study examines how extracts from an Amazonian plant affect worm development, reproduction, and lifespan, revealing differences in toxicity and antioxidant effects.
Contribution
The study reveals distinct biological effects of leaf and fruit pericarp extracts of Sapindus saponaria on C. elegans, including antioxidant and longevity modulation.
Findings
ESL showed higher acute toxicity than ESF in C. elegans.
ESL extended lifespan and upregulated antioxidant genes more effectively than ESF.
Both extracts impaired reproduction and survival in a dose-dependent manner.
Abstract
Sapindus saponaria L. (S. saponaria), popularly known as “saboeiro” or “monkey soap,” is traditionally used in South America for inflammatory, infectious, and dermatological conditions. Despite its wide use, toxicological data remain limited, and the presence of triterpenoid saponins raises safety concerns. This study evaluated the toxicological and antioxidant effects of methanolic extracts from S. saponaria leaves (ESL) and fruit pericarp (ESF) using Caenorhabditis elegans as an in vivo model. ESL and ESF were chemically profiled by ESI‐MS/MS, and worms were exposed to 1, 5, and 10 mg/mL of each extract. Endpoints included lethality (LC50), survival, development, reproduction, oxidative stress resistance, lifespan, and expression of antioxidant genes (gst‐4, ctl‐1/2/3). Both extracts contained triterpenoid saponins, while glycosylated sesquiterpenes were detected only in ESF. ESL…
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FIGURE 12| Parameter | Value | |
|---|---|---|
| Source | Capillary voltage | 4.5 kV |
| Drying gas temperature | 200°C | |
| Drying gas flow | 4 L/min | |
| Nebulizing gas pressure | 2.0 bar | |
| Transfer | Funnel 1 RF | 200 vpp |
| Funnel 2 RF | 200 vpp | |
| isCID Energy | 0.0 eV | |
| Hexapole RF | 100 vpp | |
| Quadrupole | Ion energy | 8 eV |
| Low mass | 700 m/z | |
| Collision cell | Collision energy | 5 eV |
| Collision RF | 150 vpp | |
| Transfer time | 95.0 µs | |
| Pre pulse storage | 5.0 µs |
- —Coordenação de Aperfeiçoamento de Pessoal de Nível Superior10.13039/501100002322
- —Conselho Nacional de Desenvolvimento Científico e Tecnológico10.13039/501100003593
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Taxonomy
TopicsNatural product bioactivities and synthesis · Genetics, Aging, and Longevity in Model Organisms · Phytochemistry Medicinal Plant Applications
Introduction
1
The search for bioactive compounds derived from plants with therapeutic potential has intensified in recent years, driven by the growing demand for alternatives to synthetic medicines, which are often associated with adverse effects or microbial resistance [1, 2]. Among medicinal plants of pharmacological interest, Sapindus saponaria L.—commonly known as soapwort or “monkey soap”—has been traditionally used in folk medicine throughout Latin America. Belonging to the Sapindaceae family and widely distributed across tropical and subtropical biomes of the Americas, this species has been employed in the treatment of infectious and inflammatory conditions, digestive disorders, and dermatoses [3, 4, 5].
The broad therapeutic applicability of S. saponaria is mainly attributed to the presence of triterpenoid saponins and glycosylated sesquiterpenes found in its leaves and fruits. These compounds exhibit natural surfactant properties and have been associated with a range of pharmacological activities, including antimicrobial, antifungal, anti‐inflammatory, antitumour, and wound‐healing effects [6, 7].
Phytochemical studies have identified more than 30 saponins and numerous glycosylated sesquiterpene derivatives in S. saponaria extracts. The concentration and composition of these metabolites may vary depending on the plant part analysed and the stage of maturation [8, 9]. Despite their promising pharmacological profile, toxicological investigations involving S. saponaria remain limited. Although saponins are recognized for their bioactivity, they may also exert concentration‐dependent toxic effects, including haemolytic, genotoxic, and larvicidal activities, depending on the biological system exposed [10, 11, 12].
Therefore, evaluating the safety profile of extracts rich in these compounds is crucial for their rational use in pharmaceutical and cosmetic applications. In this context, the nematode Caenorhabditis elegans has emerged as a relevant in vivo model for assessing biological and environmental toxicity. Its well‐characterized genome, short life cycle, ease of maintenance, and conserved metabolic and stress‐response pathways in mammals make C. elegans a powerful tool for the investigation of multiple toxicological endpoints, including lethality, survival, reproductive function, longevity, and oxidative stress [13, 14]. These attributes make it particularly suitable for the toxicological screening of natural extracts, such as those derived from S. saponaria.
The aim of this study was to investigate the phytochemical composition and potential biological effects of methanolic extracts obtained from the leaves and fruit pericarp of S. saponaria L., a plant traditionally used in folk medicine. By employing C. elegans as a model organism, we assessed extract‐associated toxicity, survival outcomes, and oxidative stress responses across different concentrations. This approach seeks to contribute to the ethnopharmacological understanding of S. saponaria, providing insights into its safety profile and potential for controlled therapeutic application.
Material and Methods
2
Material Collection and Methanolic Extract Preparation From Leaves and Fruits of S. saponaria L.
2.1
The plant material was collected in October 2023 from a rural property located in the municipality of Vale do Paraíso, Rondônia, Brazil (coordinates 10°34′41″ S, 62°08′02″ W). Taxonomic identification was performed by the João Geraldo Kuhlmann Herbarium of the Federal University of Rondônia (UNIR), where a voucher specimen is deposited under number RON26243, with registration code AD2F8E4 in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen).
Methanolic extracts were prepared from the leaves (ESL) and fruit pericarp (ESF) of S. saponaria L. Dried leaves and fruit pericarp of S. saponaria L. were processed separately and milled to a moderately coarse powder. For methanolic extraction, 200 g of plant powder (leaves or fruit pericarp) were mixed with 980 mL methanol (plant‐to‐solvent ratio 1:4.9, w/v) and kept at rest for 24 h (static maceration; single extraction cycle). After this period, the mixture was filtered through filter paper, and the filtrate was concentrated under reduced pressure using a rotary evaporator (400 mmHg, 55°C, 15 min). After rotary evaporation, the concentrates were transferred to pre‐weighed glass dishes and dried in a ventilated oven at 45°C until constant mass, defined as a mass variation ≤ 0.5% between two consecutive weighings performed 24 h apart [15].
For bioassays, dried extracts were solubilized in 2.5% (v/v) Tween 80 and homogenized. Stock solutions (“mother solutions”) were prepared at 100 mg/mL, and working concentrations of 1, 5, and 10 mg/mL were obtained by dilution. A 2.5% (v/v) Tween 80 solution, prepared and handled under identical experimental conditions, was used as the negative control.
Chemical Characterization by Electrospray Ionization Tandem Mass Spectrometry (ESI‐MS/MS)
2.2
The ESI–MS/MS analyses were performed using a high‐resolution mass spectrometer, micrOTOF‐Q III (Bruker Daltonics, Bremen, Germany), equipped with an electrospray ionization (ESI) source. Prior to ESI–MS/MS analysis, aliquots of the dried extracts (ESF and ESL) were reconstituted in HPLC‐grade methanol to a final concentration of 1.0 mg/mL (w/v). Samples were vortex‐mixed (and sonicated for 10 min), then filtered through a 0.22 µm PTFE syringe filter. The filtrates were transferred to a glass syringe and analysed by direct infusion using a syringe pump (300 µL·h^−^ ^1^). Tween 80 solutions were used only for the in vivo assays and were not analysed by MS. Samples were analysed in positive ionization mode with detection of singly charged ions ([M+H]^+^), multiply charged ions ([M+nH]^n^ ^+^), and adducts ([M+Na]^+^, [M+K]^+^, [M+NH_4_]^+^), as well as in negative ionization mode with detection of deprotonated ions ([M−H]^−^). Nitrogen was used as the drying and nebulising gas, and mass spectra were acquired over a mass‐to‐charge (m/z) range of 100–1500 at a spectral acquisition rate of 1.00 Hz. Sample infusion was carried out using a syringe pump operating at a flow rate of 300 µL·h^−^ ^1^. The mass spectrometer was calibrated using a 10 mM sodium formate solution. Putative annotation of major ions was performed by combining: (i) high‐resolution accurate mass and expected adduct formation ([M+Na]^+^/[M+K]^+^/[M+H]^+^ in positive mode; [M−H]^−^ in negative mode), and (ii) MS/MS fragmentation, prioritizing diagnostic neutral losses consistent with glycosidic moieties and the characteristic 42 Da spacing indicative of sequential acetylation in the sesquiterpene series. Assignments are therefore reported as putative/tentative in the absence of authentic reference standards for all constituents. And data were processed using DataAnalysis software version 4.3 (Bruker Daltonics, Bremen, Germany). Other experimental conditions are listed in Table 1.
C. elegans—Strain and Maintenance
2.3
C. elegans strains and Escherichia coli OP50 were obtained from the Caenorhabditis Genetics Center (CGC; University of Minnesota, Minneapolis, MN, USA). The Bristol N2 strain was used as the wild‐type control. The transgenic strain CL2166, dvIs19[pAF15(gst‐4b::GFP::NLS*)], which carries a GFP reporter under the control of the gst‐4 (glutathione S‐transferase‐4) promoter, was used to visualize gst‐4 expression. In addition, the GA800 strain, characterized by the presence of the transgene wuIs151[ctl‐1(+)+ ctl‐2(+)+ ctl‐3(+) + myo‐2p::GFP]*, contains a GFP reporter under the control of the myo‐2 promoter, allowing in vivo observation of the expression of the integrated antioxidant enzymes ctl‐1(+), ctl‐2(+), and ctl‐3(+). Nematodes were maintained and analysed at 20°C on nematode growth medium (NGM) agar plates supplemented with E. coli OP50. All worms used in the study were synchronized from eggs obtained by standard sodium hypochlorite treatment of gravid hermaphrodites [16, 17]. Worms were randomly allocated to treatment groups, and outcome assessments (e.g. survival counts and pharyngeal pumping frequency) were performed by independent evaluators blinded to the treatment conditions whenever possible. The in vivo experiments were conducted under authorization number 40462 granted by the Research Commission of the Federal University of Rio Grande do Sul (COMPESQ/UFRGS). As the study was performed using an invertebrate animal model, approval by an animal research ethics committee was not required, in accordance with applicable legislation.
Median Lethal Concentration (LC50)
2.4
Lethality assays were performed in 96‐well microplates with a final volume of 300 µL of liquid K medium per well, supplemented with food (5 µL of heat‐inactivated E. coli suspension). Approximately 20 synchronized C. elegans nematodes at the L1 (juvenile) stage were transferred to each well containing serial dilutions of methanolic extracts from the fruit pericarp (ESF) and leaves (ESL) of S. saponaria at concentrations of 1, 5, and 10 mg/mL. The concentration range was defined based on preliminary toxicity screening. The negative control consisted of a 2.5% (v/v) Tween 80 solution in K medium, applied in duplicate on each plate. Plates were incubated at 20°C in the dark for 24 h. Following exposure, the numbers of live and dead nematodes were recorded by visual inspection using a dissecting microscope. Nematodes were considered dead if they failed to respond to gentle mechanical stimulation with a fine metal probe. Each assay was conducted in triplicate and repeated twice independently, following standardized acute toxicity protocols for C. elegans as previously described by Fuentes et al. (2022) [18]. LC_50_ values were calculated using linear regression analysis to estimate the concentration required to induce 50% mortality under each experimental condition.
Survival Assay
2.5
Approximately 20 C. elegans individuals at either the L1 (larval) or L4 (adult) developmental stage from the N2 strain were allocated per well in 96‐well plates containing liquid K medium (K‐Medium Plus: KCl, NaCl, CaCl_2_, MgSO_4_, and cholesterol). Nematodes were treated with different concentrations of ESF (1, 5, and 10 mg/mL) and ESL (1, 5, and 10 mg/mL), or with vehicle only (negative control), and maintained at 20°C for 24 h (short‐term exposure) and 72 h (long‐term exposure), always in the presence of food (5 µL of heat‐inactivated E. coli suspension). Worm viability was assessed using a dissecting microscope, with counts performed before exposure and after 24 and 72 h of treatment. The survival rate was calculated using the formula: (number of live worms at the end of the experiment / number of live worms at the beginning of the experiment) × 100 [19, 20].
Development Assay
2.6
For body size evaluation, wild‐type C. elegans (strain N2) were age‐synchronized, and the eggs were incubated on nematode growth medium (NGM) agar plates containing different concentrations of ESF (1, 5, and 10 mg/mL) and ESL (1, 5, and 10 mg/mL), or vehicle only (negative control), and maintained at 20°C. Plates were supplemented with E. coli OP50 as a food source. Upon reaching reproductive maturity, adult worms were collected for morphological analysis. NGM plates were rinsed with 1.0 mL M9 buffer per plate, and the suspension was transferred to tubes and centrifuged at 1000 × g for 3 min (NT825, Novatécnica, Brazil) to pellet worms. The supernatant was discarded, and the pellet was resuspended in 1.0 mL fresh M9. This washing step was repeated three times (3×) to remove residual bacteria. After the final wash, worms were resuspended in M9 and a 20 µL aliquot was mounted for imaging. A 20 µL aliquot of the worm suspension was placed on a glass slide, and live worms were anaesthetized using 0.5 M sodium azide. Photographs were taken of fifteen worms per treatment group across three independent biological replicates. Worm length was measured using ImageJ software (NIH), based on images acquired using a dissecting microscope ([20]).
Pharyngeal Pumping Assay
2.7
Pharyngeal pumping activity was evaluated following both short‐term (24 h) and long‐term (72 h) exposure to different concentrations of ESF (1, 5, and 10 mg/mL) and ESL (1, 5, and 10 mg/mL), or vehicle only (negative control), with worms maintained at 20°C. Age‐synchronized N2 worms at the L3–L4 larval stage were transferred to K medium containing the respective treatments. After the exposure period, worms were transferred to nematode growth medium (NGM) agar plates, with or without E. coli OP50, for a 30‐minute acclimatization period. Pharyngeal pumping was subsequently quantified under a light microscope (Moticam Europe, 1080INT) at 10× magnification. Pumping frequency was determined by counting the number of terminal bulb contractions over a 10‐second interval. Assessments were performed by two independent evaluators. Ten worms per treatment group were analysed in each of three independent experimental replicates (Santos et al., 2024) [21].
Reproductive Assay
2.8
To evaluate the reproductive impact of the treatments on germline integrity and metabolic activity associated with germ cell function, synchronized worms at the L1 stage were exposed to different concentrations of ESF (1, 5, and 10 mg/mL) and ESL (1, 5, and 10 mg/mL), or vehicle only (negative control), and maintained at 20°C in the presence of E. coli OP50 as a nutritional source. Following prolonged exposure, the animals were allowed to develop to adulthood. Germline reproductive output was assessed by quantifying the number of fertilized eggs retained within the uterus using a dissecting microscope. This parameter was used as an indicator of germline cell health, reproductive efficiency, and potential disruptions in germ cell metabolism and oviposition mechanisms. Data represent the mean from three independent experimental replicates, using a reproductive assay adapted from the protocol described by Alvino et al. [19] and Santos et al. [21].
Oxidative Stress Assay
2.9
In wild‐type N2 Bristol worms, high concentrations of H_2_O_2_ were used to assess oxidative stress resistance. Worms were cultured on solid medium seeded with E. coli OP50 until reaching the desired developmental stages (L1–L4). Subsequently, worms were transferred to liquid medium in 24‐well plates and treated with different concentrations of ESF (1, 5, and 10 mg/mL) and ESL (1, 5, and 10 mg/mL), or vehicle only (negative control), for 24 h. Following the 24 h pretreatment, worms were washed twice (2×) by centrifugation at 1000 × g for 3 min, each time resuspending the pellet in 1.0 mL M9 buffer before transfer to the H_2_O_2_ exposure plates. Approximately 20 ± 1 live worms per well were transferred, in duplicate, to new 24‐well plates and subsequently exposed to 5 mM H_2_O_2_ (L1 stage) or 25 mM H_2_O_2_ (L4 stage). Worms were maintained under standard conditions at 20°C in a BOD incubator. Viability was assessed hourly for 3 h under a stereomicroscope, with worms considered dead if they failed to exhibit pharyngeal pumping or respond to gentle tactile stimulation. A synthetic polymer wire was used for stimulation to avoid potential chemical interactions between metal probes and hydrogen peroxide. Three independent experiments were performed.
Quantification of Gene Expression of Gst‐4 and Ctl‐1/2/3
2.10
Age‐synchronized worms carrying inducible green fluorescent protein (GFP) reporters, CL2166 (gst‐4::GFP) and GA800 (ctl‐1/2/3::GFP), were synchronized using a hypochlorite solution and incubated at 20°C for 24 h. Transgenic worms at the L4 stage were treated with different concentrations of ESF (1, 5, and 10 mg/mL) and ESL (1, 5, and 10 mg/mL), or vehicle only (negative control), and maintained at 20°C in the presence of E. coli OP50. Twenty‐five randomly selected worms from each group were mounted onto microscope slides and anaesthetized with 0.5 mM sodium azide. Photographs were acquired using a fluorescence microscope (Olympus BX41 Fluorescence Microscope) with a 10× objective lens and a constant exposure time. Quantitative analysis was performed on three independent experiments, each conducted in triplicate, by measuring GFP fluorescence intensity using ImageJ software ([22]).
Lifespan Assay
2.11
Worms were pretreated with varying concentrations of ESF and ESL (1, 5, and 10 mg/mL), or vehicle only (negative control), and maintained at 20°C in the presence of E. coli OP50. The worms were subsequently transferred to solid nematode growth medium (NGM) agar plates seeded with E. coli OP50 as a food source and containing the corresponding treatments. Transfers were performed daily until completion of the egg‐laying phase. Live, dead, and missing worms were recorded daily, and dead worms were removed promptly to avoid inaccuracies in subsequent counts. Mortality was defined as the complete absence of movement in response to gentle tactile stimulation. Worms that escaped from the plates were censored and excluded from the data analysis. All experiments were carried out in triplicate to ensure the reliability and reproducibility of the findings (Park et al. 2017 [23]; Hou et al. 2019 [24]).
Graphical Representation and Statistical Analyses
2.12
The graphical representation and statistical analysis were performed using GraphPad Prism software (V 8.0.1) (GraphPad Software, Boston, MA, USA). All analyses were performed in GraphPad Prism (v8.0.1). Data are presented as mean ± SEM from at least three independent experiments unless otherwise stated. For continuous outcomes, normality and homoscedasticity were assessed (Shapiro–Wilk and variance testing); when assumptions were met, groups were compared by one‐way ANOVA followed by Dunnett's multiple comparisons test (vs. control/vehicle) or Tukey's test (all pairwise), as appropriate. When assumptions were not met, nonparametric testing was applied (Kruskal–Wallis with Dunn's multiple comparisons). Time‐to‐event data (lifespan and survival curves) were analysed using Kaplan–Meier survival estimates and compared with the log‐rank (Mantel–Cox) test. For multiple group comparisons in survival analyses, p values from pairwise tests were adjusted using the Holm–Bonferroni method. Statistical significance was set at p < 0.05.
Results
3
Analysis of S. saponaria L. fruit and Leaf Extract
3.1
Based Phytochemical analysis was carried out using the methanolic extract to identify the secondary metabolites present in the sample. In the spectrum, ion signals with a 42 Da difference can be identified, corresponding to an acetylation. The mass spectrum obtained in full scan mode from the fruit extract of S. saponaria L. is presented in Figure 1.
Mass spectra obtained in full scan mode of the extract from the fruit of Sapindus saponaria. 1= Saponins and 2 = Glycosylated Sesquiterpenes.
Through ESI‐MS/MS analysis, the presence of a saponin with m/z 949 [M+Na]+ was identified, which possibly corresponds to a heteroside formed by the combination of oleanolic acid–glucose–rhamnose–rhamnose (Structure 1, Figure 2), This glycosylation pattern has previously been reported for saponin present in the species (c6)
Saponin at m/z 949 [M+Na]+: a heteroside formed by the combination of oleanolic acid–glucose–rhamnose–rhamnose.
In this extract, the presence of glycosylated sesquiterpenes was also identified in the mass spectrum region of 1269–1385 m/z. Based on the results obtained, these are likely sesquiterpenes linked to two glycosidic chains composed of glucose–rhamnose–rhamnose (Structure 2, Figure 3), ionized as [M+K]+ adducts, with varying degrees of acetylation, consistent with fragmentation patterns and structural assignments previously reported by Murgu and Rodrigues‐Filho (2006) [25] and Amaral et al. [8]. Ion signals with a 42 Da difference, corresponding to acetylation, can be observed in the spectrum.
Glycosylated sesquiterpenes. R = H or R = Ac.
Analysis of the leaf extract revealed the presence of the saponin at m/z 949 [M+Na]+, the same compound identified in the fruit extract. The presence of glycosylated sesquiterpenes could not be detected. The mass spectrum obtained in full scan mode for the leaf extract is presented in Figure 4.
Full‐scan ESI(+)‐MS spectrum of S. saponaria leaf extract. The major ion at m/z 949.5222 is consistent with a putative saponin detected as [M+Na]+, tentatively assigned to an oleanolic acid–glucose–rhamnose–rhamnose heteroside based on ESI‐MS/MS.
LC50 of Methanolic Preparations of ESF and ESL
3.2
In Graph A, which refers to the fruit extract (ESF), there is a clear dose‐dependent increase in lethality, reaching approximately 65% at the highest tested concentration. The LC_50_ values obtained by linear regression were 32.76 ± 3.37 mg/mL for ESF and 10.47 ± 4.87 mg/mL for ESL, and the difference between these values was statistically significant (p < 0.05), confirming the greater acute toxicity of the leaf extract. These findings suggest that, although both extracts exhibit concentration‐dependent toxic effects in C. elegans, the leaf extract (ESL) is more toxic than the fruit extract (ESF), as evidenced by its significantly lower LC_50_ value. This difference may be attributed to variations in the phytochemical composition between the leaves and the fruit pericarp (Figure 5).
Determination of LC50 was performed by linear regression using stage 1 wild‐type C. elegans N2 strain, exposed to successively increasing concentrations of ESF (A) and ESL (B) for 24 hours. Each treatment group included approximately 180 ± 10 worms.
Concentration‐ and Time‐Dependent Toxicity of S. saponaria Methanolic Extracts on C. elegans Survival
3.3
The results presented in Figure 6 demonstrate the concentration‐ and time‐dependent effects of the methanolic extracts of S. saponaria L. (fruit—ESF; leaf—ESL) on the survival of C. elegans at both L1 and L4 developmental stages. For the ESF‐treated groups (panels A–D), survival rates significantly decreased with increasing concentrations (1, 5, and 10 mg/mL) at all time points. At the L1 stage, a moderate reduction in survival was observed after 24 h (A), which became more pronounced after 72 h (B), especially at 5 and 10 mg/mL. Similarly, at the L4 stage, survival was significantly reduced at all concentrations and both exposure times (C and D), with the 10 mg/mL treatment after 72 h (D) showing the most substantial effect, suggesting cumulative toxicity over time. In the ESL‐treated groups (panels E–H), the survival impairment was more pronounced than in the ESF‐treated worms. Particularly in panel F (L1 at 72 h), survival dropped dramatically, reaching near‐total lethality at 10 mg/mL. Likewise, panel H (L4 at 72 h) showed substantial reductions in survival at higher concentrations. Even at the lowest concentration (1 mg/mL), significant differences were observed at several time points (e.g., E and G), indicating a higher toxic potential of the leaf extract compared to the fruit extract. Altogether, these data suggest that both ESF and ESL extracts reduce worm survival in a concentration‐ and time‐dependent manner, with ESL exerting more potent toxic effects, particularly after prolonged exposure.
Effect of S. saponaria L. methanolic extracts on worm (wild‐type N2 strain) survival at short and long exposure. ESF: (A) L1 stage at 24 h, (B) L1 stage at 72 h, (C) L4 stage at 24 h, and (D) L4 stage at 72 h. ESL: (E) L1 stage at 24 h, (F) L1 stage at 72 h, (G) L4 stage at 24 h and (H) L4 stage at 72 h. One‐way ANOVA followed by Tukey's post hoc test was used for statistical analysis, and results are expressed as means ± standard error of the mean (SEM). Asterisk () indicates significant difference compared to the control group at p ≤ 0.05. N = 180 ± 10 worms per treatment.*
Impact of ESF and ESL Extracts on Development and Dietary Pattern
3.4
Panels A and C illustrate the effects of ESF and ESL on C. elegans body size, respectively. Both extracts induced a slight, concentration‐dependent reduction in body size compared to the control groups, with the reduction being more evident for ESL (panel C), particularly at the highest concentration (10 mg/mL). The ESF (panel A) showed a minor decrease in body size at 10 mg/mL, although the differences were less pronounced. Panels B and D depict the pharyngeal pumping rate, a measure of feeding behaviour, following exposure to the extracts. For ESF (panel B), the pumping rate exhibited a slight decrease with increasing concentrations, reaching the lowest level at 5 mg/mL, while the 10 mg/mL group showed a tendency towards recovery. In contrast, ESL (panel D) displayed a modest but progressive decrease in pumping rate at 1 and 5 mg/mL, followed by a notable increase at 10 mg/mL, indicating a potentially complex, biphasic response to the leaf extract. None of the observed variations resulted in statistically significant differences when compared to the control group (all p > 0.05), indicating that the extracts did not significantly interfere with body size or pharyngeal pumping (Figure 7).
Effects of S. saponaria L. methanolic extracts on C. elegans body size and pharyngeal pumping rate. Panels A and B show results for the ESF, while panels C and D depict results for the ESL. Body size was measured in millimetres (mm) and pharyngeal pumping rate in pumps per 10 s. Worms were exposed to increasing concentrations of extracts (1, 5, and 10 mg/mL), with Control and Tween‐treated groups as references. One‐way ANOVA, followed by post hoc Tukey's test and expressed as means ± SEM. N = 45 worms per treatment.
Impact of ESF and ESL Extracts on Number of Fertilized Eggs Within the Uterus in C. elegans
3.5
The bar graphs (Figure 8) illustrate the effect of different concentrations of ESF (Panel A) and ESL (Panel B) extracts on the number of fertilized eggs within the uterus per worm in C. elegans. In Panel A, representing ESF treatment, the number of fertilized eggs within the uterus remains relatively stable at the 1 mg/mL concentration, comparable to control and Tween groups. However, a slight but statistically significant reduction in the number of fertilized eggs within the uterus is observed at 5 and 10 mg/mL concentrations (p < 0.05), indicating a subtle reproductive impact at higher doses. In Panel B, illustrating ESL treatment, number of fertilized eggs within the uterus shows a more pronounced and statistically significant decrease (p < 0.05) beginning at 1 mg/mL, with further reductions at 5 and 10 mg/mL concentrations. This suggests that ESL has a stronger inhibitory effect on reproductive output compared to ESF. Overall, the data indicate that both ESF and ESL extracts affect reproductive efficiency in C. elegans, with ESL exhibiting a more marked concentration‐dependent suppression of number of fertilized eggs within the uterus, potentially reflecting greater impacts on germline integrity or germ cell metabolism.
Effect of S. saponaria methanolic extracts on number of fertilized eggs within the uterus per worm in C. elegans. One‐way ANOVA, followed by post hoc Tukey's test and expressed as means ± SEM. N = 45 worms per treatment.
Stage‐Dependent Antioxidant Effects of S. saponaria Fruit and Leaf Extracts in C. elegans
3.6
The results demonstrate that S. saponaria extracts exert stage‐ and concentration‐dependent protective effects against oxidative stress in C. elegans. In panel (A), L1‐stage worms treated with ESF exhibited protective effects at concentrations of 1 and 5 mg/mL; however, survival decreased at 10 mg/mL, particularly following prolonged exposure to H_2_O_2_. In panel (B), L4‐stage worms treated with ESF showed a consistent and significant protective effect at all tested concentrations, maintaining higher survival rates than the PC group even after 3 h of oxidative stress.
In contrast, panel (C) shows that L1‐stage worms treated with ESL did not exhibit significant protection, with survival rates comparable to the PC group irrespective of concentration or exposure time. However, in panel (D), L4‐stage worms treated with ESL displayed a marked increase in survival at all concentrations, indicating that the protective effect of ESL is dependent on developmental stage.
Upregulation of Ctl‐1, Ctl‐2, Ctl‐3, and Gst‐4 by S. saponaria Extracts
3.7
The results demonstrate the modulatory effects of ESF and ESL extracts on the expression of the antioxidant genes ctl‐1, ctl‐2, ctl‐3 (catalase family), and gst‐4 in C. elegans. In panel (A), treatment with ESF led to a modest increase in catalase gene expression (ctl‐1, ctl‐2, ctl‐3) across the tested concentrations, with no statistically significant differences compared with the control, indicating a mild activation of catalase‐related defences. In contrast, panel (B) shows that treatment with ESL induced a significant and concentration‐dependent increase in catalase gene expression, particularly at 5 and 10 mg/mL, suggesting that the leaf extract effectively stimulates catalase‐associated antioxidant defences. With respect to gst‐4 expression, panel (C) demonstrates that ESF significantly upregulated gst‐4 in a concentration‐dependent manner, with higher fluorescence intensity and gene expression observed at 5 and 10 mg/mL, indicating strong activation of the detoxification pathway. Similarly, panel (D) shows that ESL also promoted a robust induction of gst‐4, with significant increases at 5 and 10 mg/mL, as evidenced by intense fluorescence and elevated expression levels. Overall, both extracts effectively induced gst‐4 expression; however, ESL exerted a more pronounced effect on catalase genes compared with ESF. These findings suggest that the leaf extract may play a stronger role in enhancing enzymatic antioxidant defences related to H_2_O_2_ detoxification, while both extracts strongly activate phase II detoxification pathways mediated by gst‐4 (Figures 9 and 10).
*Effect of S. saponaria L. fruit (A, B) and leaf (C, D) extracts on the survival of Caenorhabditis elegans at larval stage L1 (A, C) or L4 (B, D) under H2O2‐induced oxidative stress, after 24 h pretreatment (1, 5, and 10 mg/mL) followed by exposure to H2O2 (5 mM for L1 and 25 mM for L4) for up to 3 h. Data are expressed as mean ± SEM (N = 180 ± 10 worms per treatment). Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test. p < 0.05 versus NC; #p < 0.05 versus PC.
*Effect of S. saponaria fruit (ESF) and leaf (ESL) extracts on the expression of antioxidant genes in C. elegans. Panels (A) and (B) show the fluorescence images and gene expression levels of ctl‐1, ctl‐2, and ctl‐3 after treatment with ESF (A) and ESL (B), respectively. Panels (C) and (D) display gst‐4 expression after treatment with ESF (C) and ESL (D). Fluorescence images correspond to GFP reporter expression linked to each gene promoter. Data are expressed as mean ± SEM. p < 0.05 compared to the control. Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test.
Differential Effects of S. saponaria Fruit and Leaf Extracts on the Longevity
3.8
The Kaplan–Meier survival curves shown in Figure 11 demonstrate that treatment with ESF at concentrations of 1, 5, and 10 mg/mL did not result in significant alterations in the longevity of C. elegans when compared with the control and vehicle (Tween) groups. All survival curves exhibited similar profiles, with a gradual decline in the proportion of surviving organisms over time. These findings indicate that ESF, even at the highest concentration tested, neither confers a protective effect nor extends survival in the nematodes evaluated.
Kaplan–Meier survival curves for Caenorhabditis elegans treated with S. saponaria fruit extract. Concentrations of 1, 5, and 10 mg/mL were evaluated, compared with control and vehicle (Tween) groups. No statistically significant differences in longevity were observed among the groups, indicating no effect of ESF on nematode survival. Analysis performed using Kaplan–Meier survival analysis with log‐rank (Mantel–Cox) test; Holm–Bonferroni correction for multiple comparisons.
Figure 12 depicts survival curves revealing a beneficial effect of S. saponaria leaf extract (ESL) on the lifespan of C. elegans. Kaplan–Meier survival analysis followed by the log‐rank (Mantel–Cox) test revealed that ESL at 5 and 10 mg/mL significantly increased lifespan compared to control and vehicle groups (p < 0.05), whereas ESF treatments did not differ significantly from controls (p > 0.05). By contrast, the 1 mg/mL concentration did not show statistically significant differences, suggesting that the longevity‐promoting effect of ESL occurs at intermediate to high doses.
Kaplan–Meier survival curves for C. elegans treated with S. saponaria leaf extract (ESL). Concentrations of 1, 5, and 10 mg/mL were tested, compared with control and vehicle (Tween) groups. Groups treated with 5 and 10 mg/mL exhibited a significant increase in survival over time, demonstrating the longevity‐promoting effect of ESL. Results are expressed as the percentage of survival over the days evaluated. Analysis performed using Kaplan–Meier survival analysis with log‐rank (Mantel–Cox) test; Holm–Bonferroni correction for multiple comparisons.
Discussion
4
The present study provides new toxicological and antioxidant insights into the methanolic extracts obtained from the fruit (ESF) and leaf (ESL) of S. saponaria L., a species traditionally used in South American ethnomedicine. The integrated approach combining phytochemical profiling by ESI‐MS/MS with a series of C. elegans bioassays highlighted both the bioactive potential and the safety considerations associated with these extracts.
ESI‐MS/MS characterization confirmed the presence of triterpenoid saponins and glycosylated sesquiterpenes, in agreement with previous reports [8, 9]. The dominant saponin detected in both extracts (m/z 949 [M+Na]^+^) likely corresponds to an oleanolic acid‐based heteroside, known for its surfactant and pharmacological properties [6, 26]. Notably, the leaf extract lacked detectable glycosylated sesquiterpenes, which may partly explain its distinct toxicological profile. These secondary metabolites are well documented for their dual bioactive and cytotoxic effects, depending on concentration and exposure conditions [27, 28].
Both ESF and ESL exhibited concentration‐dependent toxicity in C. elegans; however, ESL demonstrated greater potency, with an LC_50_ of 10.47 ± 4.87 mg/mL compared with 32.76 ± 3.37 mg/mL for ESF. This differential effect may be attributed to variations in chemical composition, particularly the absence of glycosylated sesquiterpenes in ESL, which could influence membrane interactions [20, 29]. These findings are consistent with earlier studies indicating that saponins, despite their therapeutic promise, can compromise cellular integrity and induce lethality at higher doses [11, 12].
Prolonged exposure (72 h) to both extracts significantly reduced worm survival, with ESL again displaying higher toxicity. Notably, in L4‐stage worms, ESL at 10 mg/mL caused near‐complete lethality, suggesting that more developed nematodes may be particularly vulnerable to metabolic interference [30]. This observation reinforces the developmental stage‐dependence of toxicological outcomes, as reported in previous studies employing C. elegans to assess phytocompound toxicity [27, 31].
A mild but concentration‐dependent reduction in body length was observed for both extracts, being more pronounced in the ESL‐treated group. Reduced body size may reflect interference with hormonal signalling pathways or protein synthesis during larval development [32]. Pharyngeal pumping also exhibited a subtle biphasic modulation, particularly in the ESL‐treated groups, which may suggest adaptive neuromuscular compensation or an early feeding stress response [33]. However, the absence of statistical significance indicates that these effects are modest and may only become evident under longer exposure periods or combined stress conditions.
The number of fertilized eggs within was markedly impaired by ESL at all tested concentrations, whereas ESF induced significant reductions only at 5 and 10 mg/mL. This suggests that leaf‐derived compounds may more strongly disrupt germline metabolism or gonadal signalling [34, 35]. Saponins have previously been linked to reproductive toxicity through disruption of cell membranes and hormone‐like signalling pathways [36], supporting our observations. Given that C. elegans reproduction is tightly coupled to energy metabolism and oxidative homeostasis [37], the pronounced reduction in fecundity may reflect broader metabolic dysregulation.
Pretreatment with ESF conferred significant protection against H_2_O_2_‐induced oxidative stress at both L1 and L4 stages, whereas ESL exerted protective effects only in L4 worms. These stage‐specific differences may be associated with differential activation of endogenous antioxidant mechanisms, as well as variations in cuticle permeability or detoxification capacity [38, 39]. The biphasic response, whereby higher concentrations attenuate protection, highlights the hormetic nature of plant extract effects and underscores the importance of dose optimization [40].
Both ESF and ESL significantly upregulated gst‐4, a marker of phase II detoxification, with ESL showing stronger induction of catalase genes (ctl‐1/2/3). This suggests that leaf extract constituents more effectively stimulate enzymatic antioxidant defences, possibly through physicochemical interactions with redox‐sensitive transcription factors such as skn‐1 [22, 41]. Upregulation of these genes is consistent with enhanced resilience to oxidative damage, as previously reported for plant‐derived polyphenols and saponins [42, 43].
Notably, ESL significantly extended the lifespan of C. elegans at 5 and 10 mg/mL, whereas ESF had no effect. This may reflect ESL's greater capacity to activate stress‐response pathways such as daf‐16 and skn‐1, which are known to regulate longevity and healthspan in C. elegans [44, 45]. The absence of a lifespan effect with ESF, despite its antioxidant activity, suggests that only a subset of stress‐protective responses translate into lifespan extension, reinforcing the multifactorial regulation of ageing [33, 46].
C. elegans has become a widely adopted experimental model for investigating the biological effects of herbal extracts and plant‐derived metabolites on conserved pathways governing stress resistance, reproduction, and longevity, owing to its genetic tractability, short lifespan, and high degree of conservation with mammalian metabolic and signalling networks. This organism has proven particularly valuable for elucidating the molecular mechanisms underlying the pleiotropic actions of phytochemicals, enabling the integrated assessment of toxicological, antioxidant, and pro‐longevity endpoints within a single experimental framework. Numerous studies have demonstrated that botanical preparations are capable of modulating oxidative stress defences and lifespan through canonical regulators such as DAF‐16/FOXO, SKN‐1/NRF2, HSF‐1, and MAPK signalling pathways. These effects are frequently associated with the activation of phase II detoxification systems, including the induction of markers such as gst‐4, as well as the upregulation of enzymatic antioxidant defences, notably catalases and superoxide dismutases (Hernández‐Cruz et al.; Li et al.; Jang et al.; Wang et al.).
Moreover, plant‐derived compounds often display hormetic behaviour in C. elegans, whereby low to moderate concentrations enhance stress tolerance and survival, while higher doses may elicit neutral or adverse effects, underscoring the importance of dose optimization in phytopharmacological studies (Hernández‐Cruz et al.; Hu et al.). Collectively, these findings reinforce C. elegans as a robust and integrative platform for the functional evaluation of herbal products and support its utility in elucidating redox‐dependent and signalling‐mediated mechanisms by which plant metabolites influence organismal healthspan and longevity.
Conclusion
5
The data obtained in this study provide valuable insights into the prospective use of S. saponaria L. methanolic extracts in pharmacological applications. Phytochemical analysis revealed a complex profile of triterpenoid saponins and glycosylated sesquiterpenes, which are likely to underlie the observed biological effects. Using C. elegans as an in vivo model, we demonstrated that both ESF and ESL extracts exert concentration‐dependent toxicological impacts, including reduced survival and reproductive output, with the leaf extract displaying greater potency.
Notably, both extracts activated oxidative stress response pathways by upregulating key antioxidant genes such as gst‐4 and ctl‐1/2/3, and conferred protection against H_2_O_2_‐induced stress. ESL, in particular, extended lifespan in a dose‐dependent manner, suggesting hormetic benefits at specific concentrations. These findings support the ethnopharmacological relevance of S. saponaria, whilst highlighting the importance of rigorous toxicological assessment to guide its safe and effective use.
Altogether, this study strengthens the rationale for the controlled use of S. saponaria derivatives in future therapeutic formulations and underscores the utility of C. elegans as a cost‐effective and predictive model for evaluating plant‐derived bioactive compounds (Supporting Information).
Author Contributions
Ana Carolina Anchieta Adriano: conceptualization, methodology, validation, investigation, data curation, writing – original draft, writing – review and editing. Péterson Alves Santos: conceptualization, methodology, validation, investigation, data curation, writing – original draft, writing – review and editing. Átila Bezerra Mira: methodology, validation, investigation. Juliana Souza Terada Nascimento: conceptualization, methodology, validation. Patrícia Pereira: conceptualization, methodology, validation, formal analysis, resources, supervision, writing – original draft, writing – review and editing. Sandro de Vargas Schons: conceptualization, funding acquisition, project administration, investigation, methodology, data curation, formal analysis, resources, supervision, validation, writing – original draft, writing – review and editing.
Funding
This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (number 88887.506777/2020–00) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) fellowships to Sandro de Vargas Schons, PhD and Patrícia Pereira, PhD.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Supporting File 1: cbdv71037‐sup‐0001‐SuppMat.docx
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