The dried stems and leaves of O. biflora (Kaulf.) C.Chr. [Lindsaeaceae] were cut, rinsed with distilled water, and air-dried for 2 days. Taxonomic identification was confirmed at the Herbarium of the Institute of Biology, University of the Philippines Diliman. The plant material was cut into strips, soaked, and stored in an airtight container until it was ready for extraction. Four solvents, namely, hexane, ethyl acetate, methanol, and an aqueous solution, were used sequentially, progressing from nonpolar to polar. The procedure followed the cold maceration method (Dharajiya et al., 2017), with modifications that included soaking at room temperature, collecting the coffee cake, and drying it at the same temperature. The filtrates were subsequently separated, and the solvents evaporated using a rotary evaporator at 37 °C. For the aqueous fraction, approximately 1 L of filtrate was obtained, aliquoted into 40 mL per plate, and subjected to a two-step freezing process: initial freezing at 4 °C for 1 h, followed by storage at −80 °C for 24–72 h prior to lyophilization. After lyophilization, approximately 1 g of dried aqueous extract was recovered. The resulting dried extracts yielded approximately 28 g of hexane extract, 10 g of ethyl acetate extract, 4 g of methanol extract, and ∼1 g of aqueous extract. Each dried extract was stored at 4 °C until use. The dried extracts (hexane, ethyl acetate, methanol, and aqueous fractions) were reconstituted in 0.5% DMSO to prepare stock solutions and further diluted to the required working concentrations prior to mixing with Escherichia coli OP50. No sterile filtration step was applied, as extracts were co-administered with live OP50.

The C. elegans strains used in this study included N2 wild type, UA57 (dat-1pgfp + dat-1pcat-2), and NL5901 (unc-54palpha-synucleinyfp). All strains were obtained from the Caenorhabditis Genetics Center (CGC), University of Minnesota, Minneapolis, MN, USA. The wild-type and transgenic strains were cultivated and maintained at 20 °C on nematode growth medium (NGM) agar plates prepared according to standard protocols (Porta-de-la-Riva et al., 2012), which were seeded with non-pathogenic E. coli OP50 as a food source. Temperature-sensitive mutants were also maintained at 20 °C. For each treatment group, 15 worms at the L4 to young adult stage were manually transferred onto individual plates. Because C. elegans is a free-living, non-parasitic nematode and E. coli OP50 is a non-pathogenic bacterial strain (Manalo and Medina, 2018), all experimental procedures were conducted in compliance with Biosafety Level 1 (BSL-1) guidelines.

Young adult worms were collected immediately after molting and transferred to fresh NGM plates seeded with E. coli OP50 to obtain populations of uniform age and developmental stage. After 1 hour of egg production, eggs were transferred to fresh NGM plates containing E. coli OP50. The emergence of synchronized larvae from the eggs was anticipated, and worms were transferred to new plates daily and maintained at 20 °C.

Age synchronization of C. elegans was achieved using a modified bleaching method (Sulston and Horvitz, 1977). Freshly prepared M9 buffer was used to wash gravid adult worms from NGM plates. The worm suspension was vortexed for 2 min, pelleted, and then resuspended in bleaching solution, followed by another 1 min vortex. The suspension was then washed three to four times with M9 buffer until the solution was clear. Eggs were incubated overnight at 20 °C to allow for hatching. The resulting L1 larvae were inoculated onto NGM plates seeded with E. coli OP50 and maintained at 20 °C. After 48 h, L4 larvae were collected, followed by young adults after an additional 36 h.

The sublethal toxicity assay was performed to evaluate the effects of O. biflora extracts (OBEs) on the survival and general fitness of C. elegans. Age-synchronized L4-stage N2 wild type, UA57, and NL5901 worms were transferred to NGM plates and treated with OBEs at concentrations of 5, 10, 50, and 100 mg/mL dissolved in 0.5% DMSO (Jiang et al., 2017; Alokda and Van Raamsdonk, 2022). Worms were transferred to fresh NGM plates every other day, and the appropriate concentration of OBE was mixed with E. coli OP50 during feeding. Survival was recorded every 12 h for 3 days. Worms not treated with OBE served as the negative control. Each treatment group consisted of 15 worms, with three independent trials (n = 45 biological replicates).

The UA57 (dat-1p::gfp + dat-1p::cat-2) C. elegans mutant strain was used to evaluate dopaminergic neuronal degeneration following exposure to four extracts of O. biflora: HOBE, EOBE, MOBE, and AOBE. Each extract was reconstituted in 0.5% DMSO and applied at a sublethal concentration of 5 mg/mL for at least 2 h at 20 °C until the worms reached the L4 stage (Manalo and Medina, 2018). After exposure, worms were washed three times with M9 buffer, transferred to fresh nematode growth medium (NGM) plates, and anesthetized using 20 mM sodium azide. Only the head region, specifically the four cephalic and two anterior deirid dopaminergic neurons, was observed using a fluorescence microscope (Evos® FL) every 24 h for three consecutive days. A minimum of n = 45 biological replicates was used, comprising three independent trials with 15 worms per group. Fluorescent images were analyzed using ImageJ v1.8.0_172 (National Institute of Health, NIH, Bethesda, MD, United States). The proportion of intact neurons was calculated along with the minimum, maximum, and average green fluorescent protein (GFP) intensity per neuron (Manalo and Medina, 2018). Results were reported as mean ± standard error of the mean (SEM). Neurons were classified as lost if GFP fluorescence was absent or if small, rounded fluorescent bodies were observed (Manalo and Medina, 2018). The overall corrected total cell fluorescence (CTCF) was calculated for 45 biological replicates (three independent trials, each with 15 worms). The variance was determined using the standard error of the means, and values at day 3 were normalized to baseline CTCF values at day 0 using the following formula: Overall CTCF after day  3 = mean   CTCF   day   3 mean   CTCF   day   0  

The overall CTCF at day 3 was normalized using the baseline CTCF of the sample at day 0. The 1 mM DOPA (L-DOPA ethyl ester, SML0091, Sigma-Aldrich, USA) served as the positive control, while E. coli OP50-fed worms served as the negative control.

Following the sublethality assay, a two-stage experimental design was implemented. In the initial stage, four O. biflora solvent extracts (HOBE, EOBE, MOBE, AOBE) were assessed at a single sublethal concentration (5 mg/mL in 0.5% DMSO) to determine which extract most effectively preserved dopaminergic neurons in UA57. The concentration of 5 mg/mL was selected as it was the highest dose that maintained ≥90% survival at 72 h across all strains and solvents, thereby ensuring maximal sensitivity for detecting neuroprotective effects while remaining within non-lethal limits. A dose–response assessment was not conducted at this stage, as the objective was to identify the extract exhibiting the most potent pharmacological neuroprotective activity rather than to estimate potency.

In the subsequent stage, the extract demonstrating the most pronounced effect was advanced to additional assays, including lifespan and α-synuclein misfolded protein assay, using three sublethal concentrations (0.05, 0.5, and 5 mg/mL) to determine the concentration range associated with pharmacological activity.

To determine whether the selected extract of O. biflora could ameliorate Parkinson’s disease (PD)-related pathology, an established transgenic C. elegans model (NL5901) expressing human α-synuclein fused to yellow fluorescent protein (YFP) was used following the standard protocol (Hughes et al., 2022). Age-synchronized eggs were transferred to nematode growth medium (NGM) plates, fed with or without selected extract (reconstituted in 0.5% DMSO), and incubated at 20 °C until they reached the L4 stage. Worms were then washed three times with M9 buffer, transferred to fresh NGM plates, and anesthetized using 20 mM sodium azide. Anesthetized worms were mounted and observed under a fluorescence microscope to visualize the α-synuclein misfolded protein in the body wall muscle. Quantification of misfolded protein was performed using ImageJ v1.51w through a semi-automated process. For consistency, the same anatomical region of the body wall muscle near the head was analyzed in each worm. Images were scaled, the background was removed, and they were processed using the watershed function to separate overlapping particles. ImageJ was used to calculate both the number of aggregates per image and the mean aggregate size in calibrated units (µm²). The resulting data were imported into GraphPad Prism v10 for statistical analysis, with results expressed as mean ± standard error of the mean (SEM) and visualized with appropriate error bars (Hughes et al., 2022).

The lifespan assay was conducted using age-synchronized C. elegans obtained via the modified bleaching method. Wild-type N2 and NL5901 strains were treated with selected O. biflora extract for the test groups, 1 mM DOPA as a positive control, and OP50 as a negative control. The survival status of each worm, categorized as alive, dead, or missing, was recorded daily until all worms had died (Masoudi et al., 2014). Worms were transferred to fresh nematode growth medium plates every other day to replenish the OP50 food source and prevent confounding from newly hatched progeny (Hughes et al., 2022). Worms were considered alive if they responded to a gentle touch with a platinum wire pick; absence of response was recorded as death. Survival data from all groups were used to generate Kaplan–Meier survival curves in GraphPad Prism version 9.2.0 (283), and results were expressed as mean lifespan ±standard error of the mean (SEM).

All functional assays used a minimum of number of biological replicates (n = 45) consisting of three independent trials with 15 worms per group. In the sublethality assay, the Kaplan-Meier analysis was performed, and all results were expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) and pairwise t-test with Hochberg correction were performed for dopaminergic neuronal loss assay, alpha synuclein assay and lifespan assay. The results of these assays were expressed as the mean ± SEM. The antioxidant assays used two-way ANOVA followed by Sidak multiple comparison tests. Data values were expressed as the mean ± SD. For the mechanosensation and locomotion assays, two-way ANOVA followed by post-hoc Tukey multiple comparison test was used. Data are presented as the mean ± SEM.

The maximum tolerable concentration of O. biflora extracts (OBEs) was evaluated in 3 C. elegans strains UA57, NL5901, and N2 using a sublethal toxicity assay as a preliminary assessment. A sublethal concentration was defined as the dose at which ≥90% of the population survived within 72 h, ensuring sufficient sample size for statistical analysis even at the survival threshold.

Four concentrations (5, 10, 50, and 100 mg/mL) of each solvent extract, hexane (HOBE), ethyl acetate (EOBE), methanol (MOBE), and aqueous (AOBE), were tested. Across all strains, 5 mg/mL consistently resulted in >90% survival (Figure 1: panel A, strain N2; panel B, strain UA57; panel C, strain NL5901). In contrast, higher concentrations reduced survival below 90%, limiting the number of viable samples for robust statistical evaluation.

For subsequent pharmacological assays, two additional sublethal dilutions were prepared from the 5 mg/mL stock: 0.5 mg/mL (10⁻¹) and 0.05 mg/mL (10⁻²). Thus, all downstream experiments were conducted at 0.05, 0.5, and 5 mg/mL. Each assay utilized a minimum of n = 45 biological replicates, comprising three independent trials with 15 worms per group. Survival data were analyzed using Kaplan–Meier survival curves, and results are presented as mean ± standard deviation (SD).

To evaluate the neuroprotective pharmacological effects of O. biflora prepared with Hexane (HOBE), ethyl acetate (EOBE), methanol (MOBE), and aqueous (AOBE), the C. elegans strain UA57 was utilized. This transgenic strain overexpresses tyrosine hydroxylase, leading to excessive dopamine production, which results in dopamine-induced neurodegeneration and DOPAL accumulation, promoting oxidative stress and dopaminergic neuron degeneration (Trist et al., 2019; Pingale and Gupta, 2021; Plotegher and Duchen, 2017). This model enabled the identification of the pharmacologically active extract for subsequent assays. Neurodegeneration was assessed by measuring the green fluorescent protein (GFP) intensity in dopaminergic neurons using a fluorescence microscope over 72 h, where a decrease in intensity from baseline indicated neurodegeneration.

Figures 2A,B show a general trend of initially high GFP intensity across all treatment groups on Day 1 compared to the negative control (OP50), followed by a gradual decline by Day 3. Figure 2A shows a grayscale fluorescent image of C. elegans dopaminergic neurons. On Day 1, all neurons appear intact in both groups. By Day 3, fluorescence intensity diminishes in both treatments; however, MOBE-treated worms retain more visible neurons than those treated with AOBE, suggesting better neuronal preservation. However, GFP intensity decreased in all groups over time, C. elegans treated with 5 mg/mL methanolic O. biflora extract (MOBE) exhibited significantly higher GFP intensity (p < 0.001) on day 3 compared to both the negative control and the positive control (1 mM DOPA) (Figure 2D).

Worms fed with OP50 as a negative control showed a progressive and significant decrease in GFP fluorescence over 3 days, indicating ongoing dopaminergic neurodegeneration. This serves as the baseline for degeneration in UA57 worms without any neuroprotective intervention. The lowest overall CTCF values were recorded in this group by day 3, confirming its role as the degenerative baseline (Figures 2C,D).

The DOPA 1 mM positive control slightly delayed the loss of dopaminergic neuron fluorescence by Day 1 compared to the negative control. However, by day 3, there was still a notable decrease in GFP intensity. Although statistically different from the OP50 group (p < 0.0001), the protective effect of DOPA was not as strong as observed in the methanolic extract treatments. This reflects DOPA’s partial neuroprotective effect, as it also suggests that dopamine is prone to both spontaneous and metal-catalyzed oxidation at its catechol group, forming reactive ortho-quinones (Masato et al., 2019). Enzymatic oxidation can also generate superoxide radicals, resulting in cellular damage. These dopamine-derived quinones can form neurotoxic metabolites, such as salsolinol, which disrupts catecholamine metabolism and induces oxidative stress and mitochondrial dysfunction (Trist et al., 2019).

Worms treated with AOBE showed a modest neuroprotective effect, with significantly higher GFP intensity than the positive and negative groups on day 3 (p < 0.001). This indicates that while AOBE slowed neurodegeneration, its effect was limited, possibly due to the lower solubility of polyphenols in water, as supported by prior phytochemical profiling studies.

As highlighted earlier, MOBE-treated worms displayed the highest retention of GFP fluorescence by Day 3, indicating the most potent neuroprotective effect among all extract types. The fluorescence levels were significantly higher than both negative and positive controls (p < 0.0001). This suggests that methanol effectively extracts polyphenols and neuroprotective metabolites from O. biflora, consistent with metabolite profiling, which shows that methanol extracts from O. biflora are richer in phenolic content.

As for EOBE and HOBE, both showed minimal protection compared to the positive control. The GFP intensity of the HOBE decreased significantly over 3 days, and by day 3, the values were marginally higher than those of OP50. This is in contrast with EOBE, which had substantially higher fluorescence on day 3 than OP50 (p < 0.001). Hexane’s limited ability to extract polar bioactive metabolites, such as polyphenols, could mediate the neuroprotective effect of the extract.

Treatment with 5 mg/mL MOBE significantly slowed dopaminergic neurodegeneration compared with the other extracts tested. The neuroprotective pharmacological effects of MOBE were demonstrated by sustained GFP fluorescence intensity in dopaminergic neurons over the 72-h observation period. Based on this, 5 mg/mL MOBE was selected for subsequent assays. A total of 45 biological replicates were analyzed, comprising three independent trials with 15 worms per group. Statistical evaluation of dopaminergic neuronal loss was performed using one-way analysis of variance (ANOVA), followed by pairwise t-tests with Hochberg correction. Results are expressed as mean ± standard error of the mean (SEM).

MOBE was selected based on its activity compared to the positive control and was tested consequently at concentrations of 0.05, 0.5, and 5 mg/mL using the C. elegans strain NL5901. This strain constitutively expresses human α-synuclein fused to yellow fluorescent protein (YFP) in the body wall muscle under the control of the unc-54 promoter. It is widely used as a pharmacological model of Parkinson’s disease (PD) because the progressive accumulation of α-synuclein misfolded protein in muscle cells mimics PD-related pathology and motor decline (Wong and Krainc, 2017; Van Ham et al., 2008; Long et al., 2022). Worms were exposed to MOBE from day 1 to day 7 of adulthood, and α-synuclein misfolded protein was visualized in the anterior head region of L4 worms. Both the number and size (µm²) of aggregates were quantified using ImageJ software, following the established methodology of Hughes et al. (2022). Untreated L4 worms typically exhibited 100–130 aggregates, each ranging from 0.23 to 2.9 µm².

Figure 3A shows a representative fluorescent image of NL5901 worms, demonstrating that treatment with 5 mg/mL MOBE markedly reduced visible α-synuclein aggregates on day 7 compared to the negative control (OP50). On day 1, all groups exhibited similar numbers of misfolded proteins (91–121) with small aggregate sizes (0.23–2.9 µm²; Figures 3B–E). This pattern reflects dynamic aggregation, in which small aggregates act as “seeds” for growth and fusion into larger, more neurotoxic aggregates during aging (Vidović and Rikalović, 2022; Volpicelli-Daley et al., 2011).

By day 7, 5 mg/mL MOBE significantly reduced both the number and size of α-synuclein aggregates compared to controls (p < 0.001 and p < 0.01; Figures 3B,C). Although aggregate size increased across all groups with age, the 5 mg/mL MOBE treatment maintained a lower average size (1.84 µm²; Figures 3F,G). Larger aggregates are known to exacerbate oxidative stress, disrupt proteostasis, and impair mitochondrial function, thereby worsening neurotoxicity (Peelaerts et al., 2015; Burre et al., 2018).

Further analysis using scatter plots (Figure 3F) confirmed that the 5 mg/mL MOBE treatment significantly reduced both the number and size of aggregates compared to negative and positive controls (p < 0.001 and p < 0.01). Interestingly, the 0.05 mg/mL treatment showed fewer and smaller aggregates than the 0.5 mg/mL group (p < 0.001), suggesting a non-linear dose–response that is typical of plant-derived metabolites. At low doses (0.05 mg/mL), hormesis may occur, as mild stress activates protective pathways. In contrast, the reduced effect at 0.5 mg/mL may reflect antagonistic interactions among metabolites. Nonetheless, the 5 mg/mL MOBE consistently produced the most potent pharmacological effect, markedly reducing α-synuclein misfolded protein burden and slowing plaque development.

A minimum of 45 biological replicates was analyzed, consisting of three independent trials with 15 worms per group. Data were evaluated using one-way ANOVA followed by pairwise t-tests with Hochberg correction. Results are expressed as mean ± SEM. PERMANOVA was applied to aggregate number and size, as it accounts for multivariate dispersion across treatments.

To evaluate the pharmacological effects of MOBE in vivo, lifespan assays were conducted using C. elegans. Both N2 wild-type and NL5901 strains were exposed to MOBE at concentrations of 0.05, 0.5, and 5 mg/mL starting from the L4 stage. A concentration-dependent trend was observed, with a significant extension of lifespan at 5 mg/mL (Figures 4, 5). At this concentration, MOBE increased the mean lifespan of N2 worms by 13.61% compared to 9.26% in the untreated control (Figure 4A), while NL5901 worms exhibited a 13.85% increase compared to 8.37% in the untreated control (Figure 5A). Survival analysis further showed that, at 50% survivability, MOBE-treated N2 and NL5901 worms survived until days 15 and 16, respectively, whereas the untreated controls declined earlier (Figures 4B, 5B) (Hughes et al., 2022). These findings demonstrate that MOBE exerts pharmacological effects in vivo, significantly prolonging lifespan in both wild-type and Parkinson’s disease model C. elegans.

To functionally validate the neuroprotective potential of MOBE observed in the dopaminergic neuronal loss assay, locomotor activity and mechanosensory responses were assessed in the transgenic C. elegans strain UA57. Mechanosensory integrity was evaluated using tactile and vibratory stimuli, including plate tap, harsh touch, nose touch, and gentle head touch (Figures 6A–D). In the negative control (OP50-fed worms), responses declined sharply by Day 1 and remained low through Day 3. DOPA-treated UA57 exhibited comparable or slightly greater declines.

MOBE at 5 mg/mL significantly attenuated this sensory decline. For plate tap and harsh touch assays, MOBE-treated animals maintained higher adjusted response percentages throughout the experiment (p < 0.0001 vs. OP50, Figures 6A,B). Nose touch and gentle head touch responses were also significantly preserved (Figures 6C,D).

The effects of MOBE on locomotor behaviors were examined in the UA57 C. elegans PD model. In untreated UA57 worms (negative controls), motor activity declined progressively over 3 days, with significant reductions in body bends (Figure 7A), total and long reversals (Figures 7D,E), and a markedly suppressed frequency of omega turns (Figure 7C). Treatment with the positive control accelerated this decline, consistent with dopamine-induced toxicity (Trist et al., 2019; Pingale and Gupta, 2021; Plotegher and Duchen, 2017).

In contrast, exposure to 5 mg/mL MOBE significantly preserved locomotor function. Body bends were maintained at near-baseline levels through Day 2, with only a mild decline observed by Day 3 (p < 0.0001 vs. negative control, Figure 7A). MOBE-treated worms exhibited a significant increase in short and total reversal events compared to both OP50 and DOPA groups (p < 0.0001, Figures 7B,D), alongside enhanced omega turn frequency and long reversals (Figures 7C,E). These findings demonstrate that MOBE-treated worms exhibited higher levels of both basic and complex motor outputs compared to controls in the UA57 PD model.

The MOBE was assessed for its phytochemical profile using three chemical redox-related in vitro assays: DPPH radical scavenging, ABTS cation radical decolorization, and FRAP (Rumpf et al., 2023; Benzie and Strain, 1996). In the DPPH assay (Figure 8A), MOBE demonstrated radical-quenching values ranging from 32.21% to 86.38%, while in the ABTS assay (Figure 8B), the values ranged from 29.35% to 98.56%. In both cases, the measured values were higher than those observed for the ascorbic acid reference (p ≤ 0.001). In the FRAP assay (Figure 8C), MOBE exhibited reducing capacity values ranging from 29.35% to 98.56%, compared with 6.11%–31.22% for ascorbic acid (p ≤ 0.001).

To characterize MOBE at the phytochemical level, its total phenolic content and metabolite profile were analyzed. The total phenolic content, determined by the Folin–Ciocalteu method, was 22.3 mg GAE/g (Table 1), as shown in the calibration curve (Figures 9A,B). Metabolite profiling was conducted using high-resolution ultra-performance liquid chromatography coupled with electrospray ionization/quadrupole time-of-flight mass spectrometry (HR-UPLC-ESI-QTOF-MS). Data were acquired with MassLynx 4.2 software (mass range: 50–1,500 Da), converted to ABF format with Reifycs ABF Converter, and processed in MS-DIAL version 4.9 for peak detection and identification (Figure 9B). Peak annotations corresponded to the most intense ions at each retention time, representing distinct metabolites.

Eight metabolites were identified in MOBE, matched to reference libraries in MS-DIAL, with the following retention times and m/z values: 1,4-dihydroxyanthraquinone (1.720 min, 241.0501), flavonoid 8-C glycosides (2.001 min, 595.1670), 2-O-rhamnosylvitexin (2.052 min, 579.1715), khellin (2.102 min, 283.0590), isovitexin (2.178 min, 433.1134), apigenin-8-C-glucoside (2.203 min, 433.1134), benzoic acid (2.254 min, 123.0424), and pterosin G (4.235 min, 235.1337).