The plant species W. ecuadoriensis was collected in Guayaquil, Guayas province, Ecuador (2°08’42.7”S 79°56’49.6”W) in October 2023. The species was identified by biologist Xavier Cornejo from the Faculty of Natural Sciences of the University of Guayaquil. A reference specimen (CIBE057) was deposited in the Herbarium of the Centro de Investigaciones Biotecnológicas del Ecuador, Guayaquil-Ecuador.

10 g of dried and powdered leaves of W. ecuadoriensis were macerated with methanol (three times with 100 mL each) under constant shaking using an orbital shaker (150 rpm) at 25°C for 24 h. Each extract was filtered through Whatman No. 1 filter paper and the solutions were concentrated under reduced pressure at 40°C to obtain the methanolic extract. Then, 2.9 g of the methanolic extract was suspended in 50 mL of a methanol-water mixture (1:3) and partitioned with n-hexane (HF), dichloromethane (DMF), ethyl acetate (EF), and a residual aqueous fraction (AqF). The extracts and subfractions were stored at -20°C until required for analysis.

Total phenolic content was determined according to the Folin-Ciocalteu (FC) assay (Ghareeb et al., 2017). 20 µL of the sample was mixed with 100 µL FC reagent (1:10 v/v) and 80 µL of a Na₂CO₃ solution (7.5%), incubated for 60 minutes at room temperature, and the absorbance of the resulting blue solution was measured at 760 nm in a microplate reader (Biotek Synergy HTX, Vermont, USA). The results of the total phenol content are expressed in mg of gallic acid equivalent per gram of dry extract (mg GAE/g DE). All measurements were carried out in triplicate.

Total flavonoids were determined using the aluminum chloride method (Woster, 2003). 100 µL of sample was mixed with 100 µL of 2% AlCl₃ solution in ethanol. After 60 minutes at room temperature, absorbance at 420 nm was measured using a microplate reader (Biotek Synergy HTX, Vermont, USA). The results of the flavonoid content expressed in mg of quercetin equivalent per gram of dry extract (mg QE/g DE).

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitions were performed in vitro according to the method of (Barrientos et al., 2023). The enzymes were dissolved in Tris-HCl buffer (50 mM, pH 8.0) and 5-dithiol-bis(2-nitrobenzoic) acid (DTNB) was prepared in buffer. W. ecuadoriensis fractions were prepared at a concentration of 20 mg per milliliter in buffer. 25 µL of the sample was mixed with 125 µL of DTNB and 25 µL of the enzyme (AChE 0.3 U/mL), incubated for 15 min at 37°C, then the substrates acetyl thiocholine iodide (15 mM) and butyryl thiocholine chloride (15 mM) were added, as appropriate, and the absorbance was measured at 412 nm at 37°C in a microplate reader (BioTek Instrument, Inc., Winooski, VT, USA). The results were expressed as IC₅₀ values (μg/mL). Galantamine was used as a positive control.

To determine the inhibitory activity of α-glucosidase, 600 μL of phosphate buffer (100 mM pH 6.9) was added with 250 μL of p-nitrophenyl-α-d-glucopyranoside (5 mM), 100 μL of sample (EF/AqF from W. ecuadoriensis) and incubated at 37°C for 5 min. After that, 50 μL of 0.5 U/mL α-glucosidase enzyme solution was added to start the reaction. After 15 min at 37°C, 1000 μL of Na₂CO₃ (200 mM) was added. The absorbance was measured at 400 nm (Biotek Synergy HTX, Vermont, USA). The results were expressed as IC₅₀ values (μg/mL). Acarbose was used as a positive control (Coral Caycho et al., 2020).

A widely accepted sensitive serial dilution microplate method was used to determine the minimum inhibitory concentration (MBC) (Elisha et al., 2017). Overnight bacterial cultures were adjusted to McFarland standard 1, equivalent to 3.0 x 10⁸ CFU/mL (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa). The dried extract and subfractions were dissolved in 12.5% DMSO at a concentration of 25 mg/mL and 100 µL was added to the first well of a 96-well microtiter plate and serially diluted 1:1 with water. Bacterial cultures (100 µL) were added to each well. Starting with an extract concentration of 25 mg/mL, bacteria were therefore subjected to final concentrations of 6.25 to 0.05 mg/mL. Ampicillin was used as a positive control and DMSO (12.5%) as a solvent control. The highest concentration to which the bacteria were exposed was 12.5% DMSO in the first well and decreased two-fold in each subsequent well. Microplates were incubated overnight at 37°C. Finally, the solutions of the 96-well plates were subcultured in Petri dishes with 25 mL of soy agar. The MBC was defined as the lowest concentration of the extracts at which there was no sign of bacterial growth. The results are reported as mg/mL (Viteri et al., 2021). The antibiotic ampicillin was used as a positive control. For each bacteria and extract analyzed, a positive control (without plant extract) and a blank (without bacteria) were prepared.

The separation and identification of the compounds present in the W. ecuadoriensis fractions was performed on a Compact QTOF MS + Elute UHPLC system, with the software Data Analysis 4.0 (all Bruker Daltonik GmbH, Bremen, Germany). Approximately 5 mg/mL of the fraction was dissolved in methanol and filtered through a 0.2 μm PTFE membrane and 3 µL was injected into the equipment. They were then measured in the chromatographic system consisting of a column temperature of 40°C; flow rate of 0.4 mL/min, mobile phase H₂O + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B), and elution gradient, 0–0.5 min (12% B), 0.5–11 min (1–99% B), 11–14 min (99% B), and 14–16 min (12% B). Mass spectrometry conditions: electrospray ionization (ESI) source, scanning range 50–1300 m/z, the fragmentation pattern was obtained using the spectral libraries of the MassBank of North America (MoNA), obtained from https://mona.fiehnlab.ucdavis.edu/downloads.

All the assays were performed in triplicate and represented as median ± standard deviation (SD) using Microsoft Excel software (Microsoft 365, Microsoft Corporation, Redmond, WA, USA). Statistical significance between groups was set at p < 0.05 and determined by one-way ANOVA with Tukey’s post hoc test using the commercial software Minitab 19.

The total yield of the subfractions was performed after methanolic extraction and fractionation with n-hexane, dichloromethane, and ethyl acetate, resulting in four subfractions (hexane fraction, dichloromethane fraction, ethyl acetate fraction, and aqueous fraction). The results indicated that the methanolic extract (ME) yielded of 29.0%, followed by the aqueous fraction (AqF) which reached 15.5%. The dichloromethane fraction (DMF) had an intermediate yield of 8.5%, while the hexane fraction (HF) showed a much lower yield of 3.8%. Finally, the ethyl acetate fraction (EF) presented the lowest yield with only 1.0%.

The TPC and TFC of the extract and leaf fractions of W. ecuadoriensis were examined, and the results are presented in Table 1 . The phenolic content was highest for the ethyl acetate fraction with 357.47 ± 12.78 mg GAE/g of dry extract. The flavonoid content was highest for the ethyl acetate fraction with 48.93 ± 6.32 mg QE/g of dry extract. Although this is the first report on W. ecuadoriensis, scientific information on the genus Wigandia and family Namaceae is scarce. However, there are some previous studies on other plants of the order Boraginales. The content of phenols and flavonoids in the ethanolic extract of Eriodictyon californicum leaves has been reported (Richards and Chaurasia, 2020). The authors report this species as a promising nutraceutical due to its healing properties against oxidative stress. Our results were superior to those reported for the species Symphytum officinale and Anchusa ochroleuca, with phenolic and flavonoid contents between 5.39-125.50 mg GAE/g of extract and 0.11-36.58 mg QE/g of extract, respectively (Trifan et al., 2021). In other species such as Symphytum anatolicum and Cynoglottis barrelieri the phenolic content was 32.7 and 52.8 mg GAE/g extract, respectively (Varvouni et al., 2020). In another study, it was determined that the phenolic and flavonoid content in the methanol extract of the Onosma ambigens species was lower with values of 51.19 mg GAE/g of extract and 45.39 mg QEs/g of extract, respectively (Sarikurkcu et al., 2020).

The antioxidant activity of the extract and subfractions was analyzed by DPPH, ABTS, and FRAP methods and are presented in Table 2 . These methods are widely used due to their simplicity, sensitivity, and ability to provide comparative antioxidant capacities of various extracts and compounds (Tabart et al., 2009).

The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay measures the ability of antioxidants to scavenge free radicals by monitoring the color change from purple to yellow as the DPPH radical is reduced (Baliyan et al., 2022). The results of this assay ranged from 157.54 ± 0.49 to 187.32 ± 0.56 µmol TE/g of dry extract. W. ecuadoriensis ethyl acetate fraction scavenged DPPH in a concentration-dependent way with an IC₅₀ of 17.66 µg/mL ( Figure 2A ). The hexane fraction had the highest value (IC₅₀: 97.30 µg/mL). The antioxidant activity (IC₅₀) decreased in descending order: EF > ME > AqF > DMF > HF. According to (Setha et al., 2013) IC₅₀ values ​​< 50 µg/mL are considered potent antioxidants. A study reported that the extract of the Eriodictyon californicum species showed a 93.39% inhibition of DPPH radicals at a concentration of 1.0 mg/mL (Richards and Chaurasia, 2020), similar to those obtained in our study evaluated at the same concentration (ME: 85.97%, EF: 76.12%, DMF: 81.78%, EF: 88.63%, AqF: 89.30%). Likewise, a study reported that the extract of the polar aerial part of S. officinale showed a DPPH radical scavenging activity similar to our study (138.41 µmol TE/g) (Trifan et al., 2021). In Cordia gilletii, an IC₅₀ between 3.2 - 83.5 µg/mL is reported in different extracts (Okusa et al., 2007).

The ABTS, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay involves the generation of a blue-green ABTS radical cation, which is reduced by the antioxidants present in the sample, resulting in a decrease in absorbance. Similarly, the antioxidant activity evaluated against the ABTS radical, the extract, and all fractions had similar values in the range of 183.80 ± 0.37 and 184.12 ± 0.00 µmol TE/g extract. W. ecuadoriensis ethyl acetate fraction scavenged DPPH in a concentration-dependent way with an IC₅₀ of 10.31 µg/mL ( Figure 2B ). The antioxidant activity (IC₅₀) decreased in descending order: EF > ME > DMF > AqF > HF. Likewise, the extract of the polar aerial part of S. officinale showed ABTS radical scavenging activity similar to our study (205.82 µmol TE/g extract) (Trifan et al., 2021). In another work it was observed that the Trolox equivalent antioxidant capacity (TEAC= IC₅₀ Trolox/IC₅₀ of the sample) ratio was 0.013, while in our study it was 0.18, concluding the antioxidant power of the methanolic extract of W. ecuadoriensis (Sarikurkcu et al., 2020).

The FRAP (ferric reducing antioxidant power) assay quantifies the antioxidant effect by evaluating the reduction of the ferric-tripyridyltriazine complex to its ferrous form, which has an intense blue color. FRAP values ranged from 0.65 ± 0.01 to 2.87 ± 0.01 mmol Fe/g of dry extract. The species of the genus Eriodictyon presented antioxidant activity with a concentration-dependent behavior comparable to the ascorbic acid standard (Richards and Chaurasia, 2022).

All these results demonstrate that the species W. ecuadoriensis and especially the ethyl acetate fraction present the highest antioxidant activity evaluated by the DPPH, ABTS, and FRAP methods, probably due to the higher total phenolic and flavonoid content compared to the other fractions tested ( Table 1 ). In fact, the main compounds found in EF and AqF of W. ecuadoriensis, namely caffeic acids, rosmarinic acid, cirsimaritin, luteolin-7-glycoside and apigenin-8-C-(6”acetyl)-β-D-glucopyranoside ( Table 3 ), have already been reported as DPPH and ABTS scavengers. In this sense, these results indicate that the compounds present in the EF and DMF fractions have a strong capacity to neutralize free radicals, suggesting a high bioactive potential. This antioxidant activity highlights the W. ecuadoriensis species as a promising source of natural antioxidants, which may have therapeutic and preventive applications in diseases related to oxidative stress.

In the study of the antibacterial activity of the extract and subfractions of leaves of W. ecuadoriensis against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa, it was found that the crude extract and the different fractions presented varied effectiveness ( Table 4 ). The crude extract showed remarkable activity against S. aureus (1.56 mg/mL) and E. faecalis (3.13 mg/mL), being less effective against E. coli. Curiously, the hexane fraction was the most effective against S. aureus and E. faecalis (1.56 mg/ml), while the dichloromethane and ethyl acetate fractions presented limited activity. No activity was detected in the aqueous fraction. Ampicillin was used as a control, showing high effectiveness against S. aureus and E. coli. The genus Wigandia has been reported to have antimicrobial activities. The activity of three extracts (n-hexane, ethanol, and acetone) of W. caracasana leaves has been reported against the strains Streptococcus pneumoniae, S. pyogenes, E. coli, Salmonella typhi and Shigella flexneri with zones of inhibition between 6 and 12 mm (Cáceresa et al., 1993). Another species, Wigandia urens, has reported the antimicrobial activity of the ethanolic extract of leaves against strains S. aureus, E. coli, and P. aeruginosa with diameters between 13-19 mm (Rojas et al., 2003). Another study reported the antimicrobial activity of Cordia oncocalyx (Boraginaceae) with MIC values ​​<512 µg/mL (Thyalisson da Costa Silva et al., 2024). Our results were like those reported for the species Echium humile, who reported MCB values ​​between 1.56 and 12.5 mg/mL against S. aureus, 0.19 and 12.5 against E. faecalis, 1.56 and 3.12 against E. coli, using different extraction solvents (Aouadi et al., 2022). Although the extract and subfractions showed low antibacterial activity, these results are interesting, considering that these come directly from a leaf extract.

The inhibitory activity of the most polar fractions (EF and AqF) of W. ecuadoriensis leaves was determined by spectrophotometric assays against α-glucosidase and cholinesterases (AChE, BChE) ( Table 5 ). Inhibition of α-glucosidase is seen as an effective strategy for the control of obesity and diabetes (Zengin et al., 2018). This enzyme, located at the edge of the small intestine, breaks down complex carbohydrates into glucose. By inhibiting α-glucosidase, the metabolism of complex carbohydrates is slowed down, which lowers blood glucose levels (Mustikasari et al., 2024). Figure 3 shows the effect of EF and acarbose on α-glucosidase enzyme activity. Figure 3 shows that increasing the concentration of EF (10–100 µg/mL) and acarbose (10–200 µg/mL) increased the inhibition of α-glucosidase activity. At the highest concentrations, EF and acarbose (100 and 200 µg/mL) achieved inhibitions of 73.58 ± 0.36% and 59.39 ± 0.45%, respectively. The IC₅₀ values ​​for α-glucosidase inhibition were 38.44 ± 0.75 µg/mL for EF ( Figure 3A ), which is approximately five times higher than the commercial standard, acarbose (179.07 ± 1.18 µg/mL, Figure 3B ). These results indicate that the ethyl acetate (EF) fraction is more effective than acarbose in inhibiting α-glucosidase. According to (Benjamin et al., 2024) IC₅₀ values ​​lower than 50 μg/mL are indicators of a strong potential as an inhibitor of α-glucosidase activity. In this sense, it is important to highlight that our result was obtained from a fraction and not from an isolated compound, which highlights a promising bioactive potential for the species. The presence of several unpurified compounds within the fraction suggests that upon further purification, the inhibitory activity could even be enhanced, revealing individual compounds with even stronger properties. This observed enzyme inhibition could be related to the presence of phenolic compounds described in Table 1 . On the other hand, the aqueous fraction showed low inhibition, reaching only 4.75 ± 0.16% at a concentration of 2 mg/mL, therefore, its IC₅₀ value could not be determined. Several studies have investigated the inhibitory potential of α-glucosidase in species of the order Boraginales, showing promising results. Another medicinal herb that has been used for centuries to treat diabetes is Symphytum. The inhibitory effect of the whole plant extract of Symphytum anatolicum showed a potent inhibitory activity (IC₅₀: 18.28 ± 0.31 μg/mL), comparable to that exerted by acarbose (IC₅₀: 17.05 ± 0.25 μg/mL), used as a control (Kılınc et al., 2023). The methanolic extract of Echium humile presented a value of 60 µg/mL, indicating a strong effectiveness (Aouadi et al., 2022). An in vitro antidiabetic study revealed that ethyl acetate extract exhibits 60% inhibition, at a concentration of 500 μg/mL, with an IC₅₀ value of 380 μg/mL and the IC₅₀ value of standard acarbose was 250 μg/mL (Syed Akbar et al., 2023). The aqueous extract of Glandora diffusa showed a potent inhibitory effect on a-glucosidase with an IC₅₀ value of 33.3 µg/mL, almost ten times lower than that described for acarbose 300 µg/mL. These authors attribute this activity to the compounds caffeic acid and rosmarinic acid, which have been reported as inhibitors of α-glucosidase, and which are also present in our study ( Table 3 ) (Ferreres et al., 2013). According to these IC₅₀ values, the ethyl acetate fraction studied in this work seems to show an enzyme inhibition capacity comparable to that reported in previous studies.

Cholinesterase inhibitors play a crucial role in the functioning of the nervous system and are related to the treatment of Alzheimer’s disease. Over the past two decades, the search for natural products related to AChE and BChE inhibition has increased (Ortega de Oliveira et al., 2024). Figure 4 shows the inhibition of AChE and BChE enzymes in the presence of AqF and galantamine at increasing concentrations. The results revealed a dose-dependent behavior for the AChE enzyme with an IC₅₀ value of 915.98 ± 7.25 µg/mL for AqF ( Figure 4A ). Regarding the effect on BChE, Figure 4C shows that the fraction inhibits the butyrylcholinesterase enzyme depending on the concentration with an IC₅₀ value of 380.42 ± 22.10 µg/mL for AqF ( Figure 4C ). The ethyl acetate fraction did not show any inhibitory activity. These results were higher compared to the positive control galantamine, which presented an IC₅₀ of 0.53 ± 0.03 µg/mL for AChE and 5.15 ± 0.44 µg/mL for BChE, indicating that the standard drug, galantamine, is more effective in inhibiting these enzymes compared to the aqueous fraction of W. ecuadoriensis. However, this result is still encouraging, since we are evaluating a fraction and not an isolated compound. It is likely that the bioactive compounds responsible for the activity are found in low concentration within the fraction, suggesting that further purification could significantly increase the inhibitory activity. The results obtained for AChE are comparable to that reported for the methanolic extract of Wigandia urens, with an AChE inhibition of 43% evaluated at a concentration of 1 mg/mL (Ortiz et al., 2013). There is information on the traditional use of the Wigandia urens species in Guatemala for epilepsy and psychological problems (Hitziger, 2016). On the other hand, some extracts from the Boraginaceae family (S. anatolicum, S. aintabicum, Cynoglossum creticum, C. barrelieri, and Alkanna sfikasiana) have shown inhibitory effects on AChE, BChE, and α-glucosidase (Varvouni et al., 2020). Our results presented lower IC₅₀ compared to other species of Onosma trapezuntea and Onosma rigidum with IC₅₀ values of 1270 and 1180 µg/mL for AChE and 2550 and 2060 µg/mL for BChE, respectively (Kirkan et al., 2022). Another study reported that hexane, chloroform, ethyl acetate and methanol extracts of Calophyllum gracilentum, at a concentration of 1.0 mg/mL, inhibited AChE by 3.02 ± 0.998%, 12.30 ± 5.641%, 31.62 ± 2.057% and 4.61 ± 2.129%, respectively (Seruji et al., 2024). In our study, we evaluated at the same concentration reported an inhibition of 51.20 ± 0.60%. Based on these values, the fraction investigated in this study seems to be more effective in inhibiting this enzyme compared to previously reported results.

The results obtained in this study reveal that certain fractions of W. ecuadoriensis possess a strong antioxidant potential and a remarkable inhibitory activity of the enzyme α-glucosidase, suggesting that this species could be a promising source of bioactive compounds. However, extensive research should be carried out to isolate the main compounds and determine their activity, in order to understand their mechanism of action better. Furthermore, these observations provide a valuable scientific contribution to the chemical knowledge and biological properties of the plant species Wigandia ecuadoriensis.

The compounds from the ethyl acetate and aqueous fractions of the methanolic extract of W. ecuadoriensis leaves were analyzed by UHPLC-QTOF-MS. The total ion current chromatogram in negative ESI mode is shown in Figure 5 , and the tentatively detected compounds are summarized in Table 3 . The UHPLC-QTOF-MS profile revealed the presence of 39 metabolites, belonging to the classes of phenolic acids, flavonoids, fatty acyls, naphthofuran, glycerolipids, terpene, alkaloid, prenol lipids. The tentative identification was performed in Metaboscape software, a proprietary Bruker software that allows the identification of metabolites based on their mass, fragmentation pattern, and isotopic pattern, subsequently compared with the MassBank of North America (MoNA) database. These compounds include two phenolic acids (peaks 2 and 7), eleven flavonoids (peaks 3, 5, 6, 10, 18, 23, 25, 26, 27, 28 and 30), one lignan (peak 4), four fatty acyls (peaks 8, 9, 16, 22 and 38), four prenol lipids (peaks 11, 29, 32 and 39), one naphthofuran (peak 12), one glycerolipids (peak 13), two terpenes (peaks 14 and 15), and one alkaloid (peak 17), four lactones (peaks 19, 20, 21 and 24), three steroidal (peaks 31, 35 and 36), three unknown compounds (peaks 33, 34 and 37).

The compounds identified in the subfractions of W. ecuadoriensis exhibit a wide range of biological activities. Quinic acid shows antioxidant, antidiabetic, anticancer, antimicrobial, antiviral, antiaging, protective, antinociceptive, and analgesic properties (Benali et al., 2022). 3,4-Dihydroxybenzeneacetic and caffeic acids possess antioxidant activity in rat plasma (Raneva et al., 2001). Citric acid, known for its antimicrobial and antioxidant properties (Søltoft-Jensen and Hansen, 2005). Other compounds such as 3-hydroxybenzaldehyde, p-hydroxybenzoic acid, and hyperoside exhibit multiple properties, from antioxidant and anti-inflammatory to anticancer and organ protective (Rohini et al., 2013; Chen et al., 2021; Wang et al., 2022). Luteolin-7-glucoside, rosmarinic acid, and cirsimaritin have antioxidant, antitumor, anti-inflammatory, and protective activities against various diseases (Cai et al., 2020; Silva et al., 2020; De Stefano et al., 2021). Strobilactone A is known for its antifungal activity (Cohen et al., 2011), while thymol-beta-D-glucoside and dimorpholicacid have antibacterial activity (Mundt et al., 2003; Anderson et al., 2021). Cinchonina, in addition to being an antimalarial agent, has anticancer, antiobesity, anti-inflammatory, antiparasitic, antimicrobial, and antiplatelet effects (Parveen et al., 2024). Derivatives of acaranoic acid exhibit potent antifungal action against Botrytis cinerea, Septoria tritici and Pyricularia oryzae (Hussain et al., 2012). Luteolin-8-C-β-D-glucopyranoside and apigenin-8-C-(6”acetyl)-β-D-glucopyranoside stand out for their antioxidant capacity to scavenge free radicals (Simirgiotis et al., 2013). Kurilensoside G shows moderate inhibition in sea urchin sperm tests (Stonik et al., 2008). Erinacine D promotes nerve growth factor synthesis (Kawagishi et al., 1996). Tokoronin inhibits α-MSH-induced melanogenesis with low cytotoxicity (Ukiya et al., 2020). Aureosurfactin, a biosurfactant with comparable activity to rhamnolipid, surfactin and sophorolipid (Kim et al., 2016). Hydrogenoside C enhances cell viability and procollagen type I production in UVB-irradiated Hs68 cells (Shin et al., 2019).