The petiole and stem samples of M. linifera were collected in the aningal of the Mangal das Garças, along the banks of the Guajará Bay/Guamá River (coordinates: S 1.46471° and W 48.50674°), located in the city of Belém do Pará. The specimen is registered with the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen - Brazil) under registration number A91B68B, and a specimen is deposited in the João Murça Pires Herbarium of the Emílio Goeldi Museum (MG 216695).

After collection, the samples of M.linifera petiole and stem were washed in running water. Then, they were dried in an air-circulating oven at a temperature of 60°C for a period of 24 h, and after drying, they were ground in a knife mil. A mass of 100 g of petiole and stem powder was subjected to maceration extraction with ethanol as a solvent. After a 48 h extraction period, the solutions were filtered through qualitative filter paper and concentrated using a rotary evaporator Model Q344M2, with a thermostatic bath Model Q214M2, brand QUIMIS^®. Subsequently, they were dried in an oven with air circulation at 45°C to obtain the dry extracts. The resulting crude extracts were weighed and stored in a freezer at −18°C.

The chemical profile was performed using a robotic system comprising the application modules (Automatic TLC Sample 4 – ATS4) and the TLC Visualizer photodocumentation system (CAMAG - Muttenz, Switzerland). Silica gel aluminum plates F-254 60 Å from SILICYCLE (Quebec, Canada) were used as the stationary phase, and HPLC-grade solvents (TEDIA^® COMPANY–Fairfield, USA) were used as the mobile phase.

Aliquots of 50 µg of the extracts and 1 µg of each metabolite standard were applied to the chromatoplates and eluted in a glass chamber using an isocratic system of dichloromethane/methanol/formic acid (94:5:1) to assess the presence of terpenes/steroids and dichloromethane/methanol/water/formic acid (77:20:2:1) to evaluate the presence of saponins, phenolic compounds, flavonoids, and alkaloids. Subsequently, the chromatoplates were derivatized with selective developing solutions for the evaluated metabolite classes, prepared according to the methodology described by Wagner and Bladt (2001), photodocumented, and identified by comparison with the coloration of the standards used against the developing solutions. Image analysis was performed using WinCats 1.4.6 software.

The evaluation of the free radical scavenging capacity of the metabolites presents in EPML and ESML through bioautography was performed by HPTLC, using the same parameters described in the previous test and the elution system dichloromethane/methanol/water/formic acid (77:20:2:1). After elution, the chromatoplate was sprayed with a 40 mM DPPH• radical solution in methanol, stored for 60 min protected from light and oxygen, and photodocumented under visible light. Image analysis was performed using WinCats 1.4.6 software.

The RP-UHPLC-HRMS analysis were performed with the samples prepared at a concentration of 1 mg/mL in HPLC-grade methanol and filtered through a 13 mm PTFE filter with a pore size of 0.45 µm. The extracts ESML, and EPML were analyzed using a Dionex Ultimate 3000 UHPLC chromatograph (Thermo Scientific, Bremen, Germany) coupled to a Q-Exactive high-resolution spectrometer (Thermo Scientific, Bremen, Germany). A reversed phase C18 column (Syncronis, 2.1 × 50 mm, 100 Å–Thermofisher Scientific, Waltham, United States) was used as the stationary phase, which was maintained at 40°C. Solvent A was formed by 0.1% formic acid in deionized water: 5 mM ammonium formate and solvent B was formed by methanol acidified with 0.1% formic acid. The gradient started with 5% B up to 0.3 min, 10% B up to 0.5 min, 25% B up to 1 min, 90% B up to 6 min, 100% B up to 8 min, 100% B up to 9 min, 5% B up to 9.1 min and 5% B up to 11.1 min. A flow rate of 0.4 mL min-1 was used. Mass spectra were obtained in full scan and ms² by data dependent acquisition mode, in positive and negative ionization modes, with a detection range of 150–800 m/z. Data processing was performed using Xcalibur 3.0.1 software.

The fibroblast cell line L929 (ATCC) was cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco^®), 10 mM HEPES (Sigma), 2 mM L-glutamine, 1% MEM non-essential amino acids (Sigma), 100 U/mL streptomycin and 100 U/mL penicillin (Thermo Fisher), and 0.2 μg/mL amphotericin B (Vitrocell).

The fibroblasts were seeded at 2.5 × 10³ cells/well in 96 well plates (KASVI), with a total volume of 100 μL, at 24 h in a humidified incubator at 37°C with 5% CO₂. Subsequently, the ESML and EPML extracts were solubilized in DMSO and then diluted immediately before treatment in media without FBS, ensuring that the DMSO concentration did not exceed 0.01% in each treated group. In the wells with the cell monolayer, treatment with media without FBS, or with EPML or ESML, at serial concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.19 μg/mL) of each extract for up to 24, 48, and 72 h. The treatment solutions were added without removing the previous medium (with 10% FBS), then culture medium in the experimental groups during the assay contained a low concentration of FBS (5%).

Since the extracts were solubilized in DMSO, a preliminary MTT assay was performed using a concentration curve of this solvent (ranging from 1% to 0.001%) in L929 cells to determine the highest non-cytotoxic concentration that could be used in the treatment solution. The solvent was diluted in medium without SBF. This approach aimed to minimize variability related to DMSO usage.

After these time points, the medium was aspirated, and 0.5 mg/mL of MTT (Sigma) was added to the cells and incubated for 3 h at 37°C. Following incubation, the MTT solution was removed, 100 µL of DMSO (Sigma) was added to solubilize the formazan crystals, and the plate was read using a digital microplate reader, model Polaris - Celer Biotecnologia S.A. (λ = 570 nm). The control group was considered to have 100% viability. The experiment was performed in triplicate (n = 8), in three independent experiments.

The cells were seeded at a density of 2.9 × 10⁴ cells/well in 12 well plates and incubated overnight, showing 70%–80% cell confluence. A scratch was then made in the center of each well using a sterile 10 µL pipette tip, creating the scratch at a 90° angle between the pipette tip and the plate, followed by a wash to remove the detached cells. The cells were then treated with media without FBS or ESML or EPML extracts solubilized in DMSO. This solution (extract and DMSO) was diluted, immediately before treating the cells, ensuring that the DMSO concentration did not exceed 0.01% in each treated group, with media without FBS to achieve concentrations of 0.78, 0.39, and 0.19 μg/mL. The treatment solutions were added without removing the previous medium (with 10% FBS), then culture medium in the experimental groups during the assay contained a low concentration of FBS (5%). Microphotographs of all wells were taken for up to 0, 6, 12, and 24 h using the Stereologer^® software (Stereology Resource Center, Inc., version 11.0) connected to the Medilux TCM400 inverted microscope bright-field. The wounded area in the fibroblast monolayer was quantified using ImageJ^® software.

To quantify the pixel area of the microphotographs from the cell migration assay, the images were opened in the ImageJ 1.52a software and converted to 8-bit format. Then, the FFT plugin was used, where the ‘Bandpass Filter’ dialog box was opened, and the options ‘Autoscale after filtering’ and ‘Saturate image when autoscaling’ were selected. Next, the ‘Threshold’ dialog box was opened, and the ‘Dark background’ option was selected. A minimum filter was then applied and standardized to a radius of seven pixels. After applying the filters, the wound area was selected using the magic wand tool and measured.

The pixel areas were normalized and converted into percentages, with the 0 h time point considered as 100% of the wounded area. To better visualize the morphology and to delimit the injury area, hematoxylin and eosin staining was performed at the 24 h time point. For this, after 24 h of treatment, the wells containing the cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde at 37°C for 10 min under agitation. Subsequently, the wells were washed with PBS and stained with hematoxylin for 7 min under agitation. After another wash with PBS, eosin was added to the wells for 15 min under agitation. Following this period, a final wash was performed, and the wells were microphotographed. The experiment was performed in triplicate (n = 3), in three independent experiments.

The cells were seeded at a density of 8 × 10⁴ cells/well in 24 well plates and cultured overnight on coverslips pre-treated with poly-L-lysine Subsequently, the cells were then treated with media without FBS or ESML, or EPML extracts solubilized in DMSO. This solution (extract and DMSO) was diluted, immediately before treating the cells, ensuring that the DMSO concentration did not exceed 0.01% in each treated group, with media to achieve concentrations of 0.78, 0.39, and 0.19 μg/mL applied for 24 h. The BrdU marker (5-bromo-2-deoxyuridine) at 10 µM was added 2 h before fixation. The cells were stained with anti-BrdU (1:1000) (Sigma Aldrich^®) for 24 h at 4°C and then incubated overnight with Cy3 donkey anti-mouse antibody (1:500) (Jackson Immuno Research Laboratories, Inc.) for 2 h. The cells were counterstained with DAPI(1:100,000) for 20 min. Microphotographs were taken using the Axio Imager.M2 fluorescent microscope (ZEISS). The number of BrdU-positive cells was quantified using a multi-point tool of ImageJ^® 1.52a software. The experiment was performed in triplicate (n = 3), in three independent experiments.

Statistical evaluation was conducted using the One-Way ANOVA test with Bonferroni post-test for the cytotoxicity assay; the Two-Way ANOVA test with Tukey post-test for migration analysis; and the One-Way ANOVA with Tukey post-test for cell proliferation assessment. All statistical analyses were performed using GraphPad Prism six software, version 6.01. A significance level of p < 0.05 was considered for the applied tests.

The analyses performed by HPTLC allowed for the characterization of EPML and ESML regarding the presence of several important secondary metabolites. In the chromatogram presented in Figure 2A, it was possible to identify the presence of terpenes/steroids in both extracts, characterized by the formation of purple bands at retention factors (Rf) of 0.10, 0.38, and 0.72 after spraying with VAS solution (1% (m/v) ethanolic solution of vanillin and 10% sulfuric acid), as observed in the β-amyrin (β-a) standard, as well as the similarity between the extracts in their terpene/steroid composition.

In the chromatogram shown in Figure 2B, the presence of saponins in the extracts was identified, characterized by the formation of faint purple bands at Rf values of 0.27 for ESML, and 0.19, 0.47, and 0.51 for the petiole extract after spraying with VAS solution, as observed in the Pulsatilla A. (PA) standard. Additionally, the presence of phenolic compounds in the extracts was also identified (Figure 2C), characterized by the formation of reddish-brown bands after spraying with a 0.5% (m/v) aqueous solution of Fast Blue Salt (FBS), followed by a 10% (m/v) ethanolic sodium hydroxide solution, at Rf values of 0.70, and 0.90 for the stem extract and Rf values of 0.05, 0.11, 0.18, 0.70, 0.78, and 0.90 for the petiole extract, as observed for the Rutin (Rut) standard.

It is also noteworthy, based on the intensity of the formed bands, that the petiole extract exhibits a higher presence of phenolics than the stem extract. The methods used did not allow for the identification of flavonoids and alkaloids in either extract (Figures 2D, E). Based on these results, three lower concentrations of each extract were selected for further assays.

The analysis of free radical scavenging of EPML and ESML was also conducted through bioautography using the DPPH• radical, characterized by the formation of yellow bands corresponding to the neutralization of the radical at Rf values of 0.70 and 0.90 for EPML and at Rf values of 0.27, 0.70, 0.78, and 0.90 for ESML (Figure 2F), as observed for the Rut standard (Figure 2C).

The DPPH radical assay is a test for analyzing the phytochemical profile of extracts; however, it does not reflect the interactions of ESML and EPML with biological systems. For a more comprehensive understanding, additional and broader assays in in vitro and/or in vivo models are necessary.

The samples EPML and ESML were analyzed by RP-UHPLC-HRMS allowing the detection of several substances. Exact mass and fragmentation data were compared with previous published information from the Araceae family. Combining the RP-UHPLC retention times information at the chromatograms and mass spectrometry data, peaks corresponding to sugars, fatty acids, phenolic acids, flavonoids, and anthocyanins were annotated and putatively identified in the extracts ESML, and EPML. Among the substances detected in ESML, the most abundant components were gallic acid, pinellic acid, suberic acid, coriolic acid, p-coumaric acid, and the isomers schaftoside or isoschaftoside (Figure 3). EPML was characterized by a higher number of detected ions. In EPML, the most abundant detected substances were gallic acid, caffeic acid, p-coumaric acid, 4-hydroxycinnamic acid methyl ester, pinellic acid, coriolic acid, suberic acid, sucrose, quercetin, isorhamnetin, and rutin (Figure 3). Additionally, isomers with the same exact mass as the m/z 447.09402 ion, which could correspond to structures such as quercitrin, orientin, isoorientin, luteolin-7-glucoside, kaempferol 3-O-glucoside, apigenin O-pentoside, or cyanidin 3-glucoside, were detected. Other identified isomers included m/z 477.10419 ions corresponding to isorhamnetin 3-galactoside or petunidin 3-O-glucoside; m/z 563.14142 ions corresponding to schaftoside ou isoschaftoside; and m/z 609.14679 ions corresponding to delphinidin 3-rutinoside or cyanidin 3-gentiobioside.

The structures detected in M.linifera that have previously been reported within the Araceae botanical family include p-coumaric acid, pinellic acid, schaftoside, isoschaftoside, 4-hydroxycinnamic acid methyl ester, caffeic acid, quercetin, isorhamnetin, quercitrin, orientin, isoorientin, luteolin-7-glucoside, kaempferol 3-O-glucoside, apigenin O-pentoside, cyanidin 3-glucoside, isorhamnetin 3-galactoside, petunidin 3-O-glucoside, delphinidin 3-rutinoside, cyanidin 3-gentiobioside, and rutin (Williams, 1981; Nagai et al., 2002; Su et al., 2013; Wu et al., 2009; Fawzi Mahomoodally et al., 2023; Rechek et al., 2023).

The exposure of fibroblast cells to the ESML and EPML extracts induced an increase in viability, and did not promote cellular cytotoxicity at low concentrations.

It was demonstrated that the highest concentration of DMSO in a treatment solution for L929 cells that does not affect cell viability for up to 72 h is 0.01% (Supplementary Figure S1). Therefore, all treatment solutions used in the assays conducted in this study contained a maximum of 0.01% DMSO.

The cytotoxicity results demonstrated that after 24 h of treatment with EPML, there was a reduction in viability only at the highest concentration used (100 μg/mL) compared to the control group (91.39% ± 6.11%) (Figure 4D). The other concentrations of EPML and all concentrations of ESML did not show a loss of cell viability (Figures 4A, D).

At concentrations of 3.125, 1.56, 0.78, 0.39, and 0.19 μg/mL of ESML, there was a significant increase in cell viability compared to the control, with viabilities of 100.40% ± 8.54%, 102.4% ± 9.19%, 103.90% ± 10.76%, 106% ± 9.51%, and 104.90% ± 12.22%, respectively (Figure 4A).

At the 48, and 72 h time points, there was no decrease in cell viability at any concentration of ESML and EPML used (Figures 4B, C, E, F) compared to control. At the 48 h time point, there was a significant increase in viability at the concentration of 0.19 μg/mL for both ESML and EPML compared to control, with viabilities of 107.40% ± 7.86%, and 111.70% ± 8.01%, respectively (Figures 4B, E). At the 72 h time point, a significant increase was observed at the concentration of 0.19 μg/mL of ESML compared to the control, with viability of 113.6% ± 6.05% (Figure 4C).

The scratch assay demonstrated an increase in cell migration, indicated by a significant reduction in the area of the lesion created in the cell monolayer when treated with ESML and EPML extracts. This wound closure-inducing activity suggests that these extracts may be enhancing cellular motility.

The lesions in the cell monolayer treated with ESML extract at a concentration of 0.19 μg/mL at 12, and 24 h, with lesion areas of 32.29% ± 16.62%, and 10.15% ± 7.35%, respectively. Additionally, there was a significant reduction in the lesion area when treated with the concentration of 0.39 μg/mL of ESML at the 24 h time point, showing a lesion area of 10.67% ± 7.94% (Figure 5).

The 0.78 μg/mL concentration of the ESML extract did not accelerate wound closure in vitro compared to the control group at the 6 h, 12 h, and 24 h treatment time. Furthermore, the ESML extract at a concentration of 0.39 μg/mL also did not accelerate wound closure in vitro compared to the control group at the 6 h, and 12 h treatment times. Treatment with ESML at a concentration of 0.19 μg/mL for 6 h did not accelerate wound closure in the cell monolayer.

The treatment of the lesion in the fibroblast monolayer with 0.19 μg/mL EMPL for 24 h demonstrated a significant reduction in the injured area, with an area of 12.40% ± 7.72% (Figure 6).

The concentrations of 0.78 μg/mL, 0.38 μg/mL, and 0.19 μg/mL of the EMPL extract showed no significant difference in wound closure in vitro compared to the control at the 6 h, and 12 h time. At 24 h time, the 0.78 μg/mL, and 0.38 μg/mL concentrations of the EMPL extract also showed no significant difference in wound closure in vitro compared to the control group.

In the Hematoxylin and Eosin staining, it is possible to observe a decrease in the lesion area, attributed to the increased migration of fibroblasts induced by treatment with 0.19, and 0.39 μg/mL of ESML for 24 h (Figures 7D, E), and 0.19 μg/mL of EPML for 24 h (Figures 8D, E).

The in vitro lesions were stained to enhance visualization of the wound areas treated for 24 h with ESML, and EPML extracts.

The extracts demonstrated wound closure in vitro, raising the hypothesis that, in addition to the ESML and EPML extracts inducing increased cell migration, they might also accelerate fibroblast proliferation. To investigate this, a cell proliferation assay was performed.

The proliferation rate induced by ESML and EPML was analyzed through BrdU immunostaining, marking the cells that entered the S phase of the cell cycle. The control group showed 19.78 ± 3.80 BrdU positive cells.

The concentrations of 0.39, and 0.19 μg/mL of ESML significantly increased the proliferation of fibronblasts compared to the control group, with 37.89 ± 7.41 and 29.78 ± 4.52 BrdU positive cells, respectively (Figure 9).

Accordingly, the group of cells exposed to the ESML extract at concentrations of 0.39 μg/mL and 0.19 μg/mL showed increased cell proliferation, with a higher number of BrdU-positive cells compared to the control group. The 0.78 μg/mL concentration of the ESML extract did not significantly promote cell proliferation compared to the control group over 24 h.

The concentration of 0.19 μg/mL of EPML also significantly induced the proliferation of L929 cells compared to the control, showing 24.89 ± 3.55 BrdU positive cells (Figure 10).

Thus, the EPML extract at a concentration of 0.19 μg/mL, after 24 h in contact with the cells, promoted increased proliferation compared to the control group, resulting in a higher number of BrdU positive cells in the group exposed to the EPML extract (0.19 μg/mL).