Celtis iguanaea Spray-Dried Extract Exhibits Low Acute Toxicity in Artemia salina

As shown in Fig. 1, a statistically significant rise in lethality was observed at 800, 1600, and 3200 μg/ml of SDCi when compared with the vehicle control. The calculated LC50 was 418.4 μg/ml (R2 = 0.82; 95% CI: 417.60–440.36). Although this LC50 value suggests toxicity at higher concentrations, the lethality was detected only at the upper limits of the tested range, which are well above the expected exposure levels for intended applications such as wound healing.

Fig. 1

Lethality of the Celtis iguanaea standardized spray-dried extract in Artemia salina larvae following acute exposure. Artificial seawater was used as the vehicle (negative control). *p < 0.01, **p < 0.01, and ***p < 0.001 indicate statistically significant differences compared to the vehicle group, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a 95% confidence interval. Results are expressed as mean ± SD (n = 20 larvae per group, in triplicate). SDCi: C. iguanaea standardized spray-dried extract

At the tested SDCi concentrations (3200, 1600, 800, 400, and 200 μg/ml), the corresponding estimated exposure levels were approximately 70.4, 35.2, 17.6, 8.8, and 4.4 ng/ml for polyphenols; 160, 80, 40, 20, and 10 ng/ml for flavonoids; and 1664, 832, 416, 208, and 104 ng/ml for phytosterols. These lethality results indicate a threshold effect, with a significant increase observed at 800 µg/ml compared to controls, but no further increase at 1600 and 3200 µg/ml. This plateau suggests that the toxic effect reaches a maximum beyond a certain concentration possibly due to saturation of the underlying toxic mechanism.

The relatively high phytosterol level at these concentrations may contribute to membrane or metabolic disruption (Wang et al. 2025), whereas polyphenols and flavonoids may induce cytotoxic effects when present at elevated concentrations (Jain et al. 2025). Lethality did not exceed 50% at any tested concentration, and no meaningful toxic effects were observed at concentrations below 800 μg/ml. Collectively, these results indicate a safety margin at lower exposure levels relevant to potential applications, with adverse effects emerging only at relatively high concentrations.

Artemia salina lethality assays are widely used in natural product research as a preliminary toxicity screening method (Ntungwe et al. 2020). In a previous study, the in vivo toxicity of a crude ethanolic extract from the stem bark of C. iguanaea, along with its hexane, chloroform, and ethyl acetate fractions was assessed using this assay yielding LC50 values above 1000 μg/ml for all samples tested (Trevisan et al. 2012).

The observed differences in lethality outcomes between studies can be attributed to variations in plant origin, collection period, and plant part used, as well as differences in processing and extraction methods, all of which can substantially alter the phytochemical composition of the tested material. For example, two triterpenes (i.e., friedelin and epifriedelinol) were isolated from the hexane fraction of the stem bark (Trevisan et al. 2012), but these compounds were not detected in the SDCi by UFLC-DAD-MS analysis (Ribeiro et al. 2025). Furthermore, the SDCi constitutes a high-added-value phytopharmaceutical intermediate that is standardized for specific bioactive constituents, which may result in relatively higher concentrations of certain compounds with toxicological potential.

Compared to other species of the same genus, SDCi was less toxic than various extracts from the fruit and root of Celtis pallida Torr. (1.2 μg/ml ≤ LC50 ≤ 217.8 μg/ml) (López et al. 2022). It is important to note that reports on Celtis species show considerable variability in pharmacological outcomes. Such variability likely reflects differences in plant parts used (e.g., fruits, roots, leaves), extraction methods, and resulting phytochemical composition, rather than indicating intrinsic contradictions in literature (Samadd et al. 2024; Touhtouh et al. 2025).

When compared to other medicinal plants from Brazilian biodiversity, SDCi was less toxic than the aqueous extract of Myracrodruon urundeuva Allemão, Anacardiaceae, stem bark (LC50 263.1 μg/ml) but more toxic than the aqueous stem bark extracts of Anadenanthera colubrina (Vell.) Brenan, Fabaceae (LC50 923.1 μg/ml), Hymenaea stilbocarpa (syn. of Hymenaea courbaril L.), Fabaceae (LC50 1050.2 μg/ml), and Copaifera langsdorffii Desf., Fabaceae (LC50 1380.8 μg/ml) (Senigalia et al. 2020).

Celtis iguanaea Spray-Dried Extract Demonstrates Low Acute Embryotoxicity in Zebrafish Toxicity Assay

As shown in Fig. 2, significant lethality was observed only at the highest SDCi concentrations (i.e., 1000 and 2000 µg/ml) when compared with the control. The calculated LC50 was 1116 µg/ml (R2 = 0.96; 95% CI: 965.7–1289). Celtis iguanaea has attracted growing interest due to its remarkable antinociceptive and anti-inflammatory activities (Moraes et al. 2022; Almeida et al. 2024; Ribeiro et al. 2025). When compared to commonly used anti-inflammatory drugs and herbal medicines in Brazil, SDCi can be considered less toxic than nimesulide (LC50 0.9 µg/ml) (Salgado et al. 2021), paracetamol (LC50 800.0 µg/ml) (Carvalho et al. 2024), cannabidiol (LC50 1.9 µg/ml) (Wei et al. 2024), indomethacin (LC50 10.6 µg/ml) (Liu et al. 2024), Curcuma longa L., Zingiberaceae (LC50 56.7 µg/ml), and Cymbopogon citratus (DC.) Stapf, Poaceae (LC50 9 µg/ml) (Alafiatayo et al. 2019). Nevertheless, we emphasize that literature comparisons should be interpreted cautiously, as differences in assay conditions and the tested sample can significantly influence LC50 outcomes (Su et al. 2021). These revisions aim to present a more balanced interpretation and avoid overstating the comparative advantage of SDCi.

Fig. 2

Acute toxicity of the Celtis iguanaea standardized spray-dried extract in zebrafish embryos. E3 embryo medium was used as the vehicle (negative control). **p < 0.01 and ***p < 0.001 indicate statistically significant differences compared to the vehicle group, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a 95% confidence interval. Results are expressed as mean ± SD (n = 20 embryos per group, in triplicate). SDCi: C. iguanaea standardized spray-dried extract

At the tested concentrations of SDCi (2000, 1000, 850, 650, 500, and 250 µg/ml), the estimated exposure levels for zebrafish embryos were approximately as follows: 44, 22, 18.7, 14.3, 11, and 5.5 ng/ml of polyphenols; 100, 50, 42.5, 32.5, 25, and 12.5 ng/ml of flavonoids; and 1040, 520, 442, 338, 260, and 130 ng/ml of phytosterols, respectively.

Although the OECD does not provide a formal toxicity classification system specifically for zebrafish embryo assays (OECD 2013), data generated from FET tests (e.g., LC50 values) can be aligned with international classification systems, such as the GHS. According to GHS guidelines for 96-h acute aquatic toxicity tests, the categories are as follows: category 1 – LC50 ≤ 1 mg/l (very toxic to aquatic life); category 2–1 < LC50 ≤ 10 mg/l (toxic to aquatic life); and category 3–10 < LC50 ≤ 100 mg/l (harmful to aquatic life) (Pan 2012). Since the LC50 determined for SDCi is 1116 mg/l, it can be classified as non-hazardous to aquatic life (> 100 mg/l). This interpretation applies solely to acute aquatic endpoints and does not replace chronic toxicity or mammalian hazard assessments.

Altogether, the acute toxicity findings indicate that SDCi exhibits a differential toxicity profile depending on the biological model, showing higher toxicity to invertebrates and comparatively lower toxicity to vertebrates. This selective toxicity warrants further investigation into its potential larvicidal activity against medically important species, such as Aedes aegypti (Diptera: Culicidae), the primary vector of arboviruses including dengue, zika, and chikungunya (de Oliveira et al. 2023a, b), which has been implicated in the most significant arboviral epidemics recorded in Brazil over the past twenty-five years (Gurgel-Gonçalves et al. 2024).

Celtis iguanaea Spray-Dried Extract Shows Promise for Wound Healing Applications

As illustrated in Fig. 3, SDCi did not induce cytotoxic effects in either WI-38 or HaCaT cell lines at any of the concentrations tested. Notably, a significant increase in cell viability was detected at the highest concentration tested (100 µg/ml; p = 0.0447), indicating a potential proliferative or metabolic stimulatory effect of SDCi at this concentration. This finding supports the hypothesis that the extract may have beneficial effects on keratinocyte function and skin homeostasis.

Fig. 3

Evaluation of cytotoxicity induced by the Celtis iguanaea standardized spray-dried extract in WI-38 (A) and HaCaT (B) cell lines after 24 h exposure, using the MTT assay. Saline solution was used as the vehicle (negative control). *p < 0.05 indicates statistically significant differences compared to the vehicle group, as determined by Student’s t- test, with a 95% confidence interval. Results are expressed as mean ± SD of three independent replicates. SDCi: C. iguanaea standardized spray-dried extract

Based on these findings, the highest concentration (100 µg/ml) was selected for subsequent functional assays, including cell adhesion and migration. Since cell migration is a critical step in re-epithelialization during wound healing (Li et al. 2024), selecting a concentration that promotes keratinocyte viability helps ensure that the observed effects are not confounded by cytotoxicity. Moreover, the enhanced metabolic activity observed at 100 µg/ml may indicate an activated phenotype conducive to wound repair, providing further rationale for its application in migration studies.

Figure 4 shows that SDCi at 100 µg/ml significantly increased the adhesion of HaCaT keratinocytes to the substrate by approximately 12% (p = 0.0142), suggesting a potential role in promoting epithelial cell–extracellular matrix interactions. Although these findings are promising, we acknowledge that additional concentrations should be evaluated in future studies to establish a complete dose–response profile. Moreover, these adhesion-related effects should be interpreted as supportive but not conclusive evidence of wound-healing potential, particularly in the absence of reproducible migration assays and in vivo confirmation. Nevertheless, the significant response observed at 100 µg/ml provides relevant preliminary insight and reinforces the hypothesis that SDCi may beneficially modulate keratinocyte behavior.

Fig. 4

Evaluation of cell adhesion induced by the Celtis iguanaea standardized spray-dried extract in HaCaT cell lines after 24 h exposure, using the matrigel assay. *p < 0.05 indicates statistically significant differences compared to the vehicle group, as determined by Student’s t-test, with a 95% confidence interval. Results are expressed as mean ± SD of three independent replicates. SDCi: C. iguanaea standardized spray-dried extract

Cell adhesion is a crucial early event in the wound healing process, as it precedes and facilitates cell migration, proliferation, and re-epithelialization (Jin et al. 2025). The ability of SDCi to enhance cell attachment may therefore reflect bioactive properties that support tissue regeneration and maintain skin barrier integrity.

This pro-adhesive effect could be attributed to the modulation of cell surface receptors or signaling pathways involved in adhesion, such as integrins and cadherins, or to structural alterations in the cytoskeleton that favor stronger cell–matrix anchorage (Hoshino et al. 2022; Yamada et al. 2023). Furthermore, the observed increase in adhesion may suggest that SDCi contains compounds capable of enhancing the deposition or interaction with extracellular matrix components, contributing to a more favorable environment for cell attachment (Zulkefli et al. 2023). Taken together, these results provide preliminary support for a potential role of SDCi in promoting skin repair, justifying further studies involving migration and wound closure assays, as well as evaluations of focal adhesion markers and adhesion-related gene expression.

This perspective gains further relevance when considered alongside previous evidence that C. iguanaea hydroalcoholic leaf extract improves systemic metabolic parameters in hypercholesterolemic rats (Zanchet et al. 2018). In that study, the extract reduced serum total and LDL cholesterol, inhibited HMG-CoA reductase, and lowered pro-inflammatory cytokines (IL-1, IL-6, TNF-κ, and IFN-γ). Additionally, it also demonstrated hypoglycemic effects, decreased disaccharidase activity, increased muscle glycogen storage, enhanced antioxidant defenses, and reduced hepatic steatosis. These metabolic improvements, particularly glycemic control, anti-inflammatory activity, and antioxidant effects, are critical determinants of wound-healing outcomes, especially in diabetic conditions (Xiang et al. 2019; Dasari et al. 2021). Consequently, future studies will focus on evaluating the wound-healing potential of SDCi in diabetic rat models, where metabolic dysfunction often impairs tissue repair.

The highest non-cytotoxic concentration tested in vitro (100 µg/ml) corresponds to an estimated dermal exposure of 210 µg/cm2, assuming 100% release from a formulation containing 7% (70 mg/g) SDCi applied at 3 mg/cm2. Such exposure levels are within or exceed the anticipated concentrations in finished products planned by our research team, suggesting a favorable safety margin for the intended use. Although a comprehensive quantitative risk assessment requires systemic absorption and toxicokinetic data (Visintin et al. 2025). These findings provide preliminary reassurance regarding safety at expected user exposure levels.

Although LC-DAD-MS analysis enabled the tentative annotation of several metabolites in SDCi, the structural information obtained was insufficient to establish definitive phytochemical–effect relationships. For the flavonoids, phenolic acids, and fatty acids detected, the data did not allow us to determine the precise sugar moieties, stereochemistry, or the position of hydroxyl substitutions, features known to critically influence biological activity. Consequently, the metabolites annotated in this study cannot yet be directly linked to the cellular responses observed. Future work employing high-resolution structural techniques (e.g., NMR, MS/MS fragmentation using authentic standards, or targeted isolation and purification strategies) will be necessary to conclusively identify these compounds and elucidate their mechanistic contributions.

In light of these unresolved phytochemical uncertainties, it is also important to recognize that a comprehensive dermal safety assessment requires additional information, including percutaneous absorption, systemic exposure estimates, and margin-of-safety (MoS) calculations. Without precise structural identification of the major constituents, toxicity prediction using in silico tools such as PredSkin (Borba et al. 2021) or the construction of MoS-based risk assessments would lack reliability. Therefore, any long-term or systemic safety inference would be premature at this stage. Future studies integrating high-resolution compound identification, percutaneous absorption assays, and subchronic dermal exposure models are essential to establish a robust toxicological and risk-assessment framework for SDCi.

Celtis iguanaea Spray-Dried Extract Does Not Induce Genotoxicity In Vitro

The safety of natural products intended for therapeutic or cosmetic applications requires careful assessment of their cytotoxic and genotoxic potential. As shown in Fig. 5, the resazurin reduction assay revealed that SDCi caused no cytotoxic effects in either Caco-2 or HT-29 cells, even at concentrations as high as 250 µg/ml, indicating maintained mitochondrial metabolic function and cell viability. These results suggest that the extract is well tolerated by human intestinal epithelial cells, supporting its biocompatibility.

Fig. 5

Evaluation of cytotoxicity induced by the Celtis iguanaea standardized spray-dried extract in Caco-2 and HT-29 cell lines after 24 h exposure, using the resazurin assay Low-glucose DMEM was used as the vehicle (negative control). No statistically significant differences were observed compared to the vehicle group, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a 95% confidence interval. Results are expressed as mean ± SD of three independent replicates. SDCi: C. iguanaea standardized spray-dried extract

To further investigate the genotoxic potential of SDCi, a single-cell gel electrophoresis (comet) assay was conducted, with the results presented in Fig. 6. Treatment with SDCi at 25, 50, and 100 μg/ml did not lead to a significant increase in DNA strand breaks, as reflected by unchanged tail moment (Fig. 6A) and tail intensity (Fig. 6B) values in both cell lines. In contrast, exposure to methyl methanesulfonate (400 µM), used as a positive control, significantly increased both parameters (p < 0.001), thereby confirming the sensitivity and validity of the assay. These findings confirm that SDCi does not induce detectable genotoxic damage under experimental conditions. Therefore, the absence of cytotoxic and genotoxic effects in two distinct epithelial cell lines reinforces the safety profile of the SDCi and supports its potential for use in formulations intended for topical or oral exposure.

Fig. 6

Tail moment (A) and intensity (B) in Caco-2 and HT-29 cells assessed by the comet assay after 24 h exposure to the Celtis iguanaea standardized spray-dried extract. Methylmethanesulfonate; Low-glucose DMEM and methylmethanesulfonate were used as the vehicle (negative control) and positive control, respectively. ***p < 0.001 indicates statistically significant differences compared to the vehicle group, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a 95% confidence interval. Results are expressed as mean ± SD of three independent replicates. SDCi: C. iguanaea standardized spray-dried extract; MMS: methylmethanesulfonate

It is important to note that the present study evaluates only acute toxicity endpoints. While the findings provide preliminary evidence supporting the acute safety of SDCi, long-term toxicological assessments (chronic and subchronic) will be required to establish its safety under repeated or prolonged topical or oral use. These studies are currently planned as part of our ongoing research efforts.

Celtis iguanaea Spray-Dried Extract Shows No Mutagenic Effects In Vitro

To complement the evaluation genotoxicity, the potential mutagenicity of SDCi was evaluated using the micronucleus assay in Caco-2 cells. As presented in Table 1, exposure to SDCi at 25, 50, and 100 μg/ml did not significantly increase the number of micronuclei compared to the vehicle-treated group, indicating no evidence of chromosomal damage or mitotic spindle dysfunction. In contrast, treatment with the positive control (methyl methanesulfonate, 264 µM) caused a marked increase in micronucleus formation (p < 0.01), confirming the assay’s sensitivity. These results reinforce the non-mutagenic nature of the SDCi and align with the findings from the resazurin and comet assays, further supporting the in vitro safety of the extract.

Table 1 Micronucleus assay in Caco-2 cells after 20 h exposure to Celtis iguanaea standardized spray-dried extract

Ultrapure water and methyl methanesulfonate were used as the vehicle (negative control) and positive control, respectively. **p-value < 0.01 indicates statistically significant differences compared to the vehicle group, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a 95% confidence interval. Results are expressed as mean ± SD of three independent replicates. SDCi: C. iguanaea standardized spray-dried extract; MMS: methylmethanesulfonate; CBPI: Cytokinesis-Block Proliferation Index.

Celtis iguanaea Spray-Dried Extract Exhibits No In Vitro Antibacterial or Antifungal Activities

Table 2 summarizes the results of the in vitro antifungal and antibacterial activity of SDCi. At the concentrations tested (up to 1024 µg/ml), SDCi showed no inhibitory effect against the evaluated yeast and bacterial strains. It is worth noting that the extract also showed no inhibitory effect against the included multidrug-resistant clinical isolates. Considering that the development of antimicrobial resistance is often accompanied by reduced virulence and impaired bacterial fitness (Cepas and Soto 2020), these findings reinforce that, even when the microorganisms may be at a physiological disadvantage, SDCi does not exhibit meaningful antibacterial activity.

Table 2 Minimum inhibitory concentration of the Celtis iguanaea standardized spray-dried extract against selected yeast and bacterial reference strains and clinical isolates

In contrast, the MIC values for nystatin ranged from 1 to 4 µg/ml for yeasts, confirming the susceptibility of the strains and validating the assay. For bacteria, ciprofloxacin exhibited MIC values ranging from 0.03125 to 1 µg/ml, consistent with BrCAST protocol standards. Regarding the clinical isolates, the MIC values obtained for colistin and meropenem were fully consistent with the BrCAST protocol standards, confirming the reliability and robustness of the reference controls used in the assays. Sterility controls for both the culture media and tested compounds indicated no contamination. Additionally, the growth control was successful for all strains, which proliferated in the absence of antimicrobial agents.

These findings align with previous studies on Celtis species, which demonstrate that antimicrobial activity varies considerably depending on the extraction method and solvent polarity. For example, methanolic extracts of C. australis L. from Morocco exhibited moderate antibacterial activity against E. coli and S. aureus (MIC 100 µg/ml), and stronger antifungal activity against C. albicans (MIC 50 µg/ml), with phenolic compounds and phytosterols identified as the major constituents (Filali-Ansari et al. 2016). Similarly, a recent study on methanolic extracts of C. integrifolia Lam. from Nigeria reported weak antibacterial activity, with MIC values of 250,000 µg/ml against E. coli and 125,000 µg/ml against S. aureus (Sa’id and Abba 2021).

In contrast, an aqueous extract of C. integrifolia obtained via reflux reported no inhibitory activity against E. coli, S. aureus, P. aeruginosa, or C. albicans, despite the presence of alkaloids, saponins, phenolics, phytosterols, tannins, and glycosides (Abah et al. 2019). Similarly, an extract of C. australis prepared with water and ethanol in Croatia exhibited limited antibacterial activity, with MIC values of 5,000 µg/ml against E. coli, and greater than 10,000 µg/ml against P. aeruginosa and S. aureus (Ota et al. 2017).

Hence, extracts obtained with less polar solvents, particularly methanol, have generally demonstrated stronger antimicrobial effects, as reported for C. australis and C. integrifolia (Filali-Ansari et al. 2016; Sa’id and Abba 2021). Conversely, highly polar solvents such as water or hydroethanolic mixtures tend to yield extracts with little or no antimicrobial activity (Ota et al. 2017; Abah et al. 2019). This trend suggests that the weak activity observed for SDCi may be partly attributable to the high polarity of the solvent used during extraction. Future investigations employing less polar solvents or increased extract concentrations may provide further insight into its antimicrobial potential.

According to one classification, antimicrobial activity is considered significant when MIC values are below 100 µg/ml, moderate between 100–625 µg/ml, and weak when exceeding 625 µg/ml (Kuete 2010). Alternatively, other authors consider the activity significant when the MIC is below 10 μg/ml, moderate between 10–100 μg/ml, and low when > 100 μg/ml (Mbaveng et al. 2015). Based on these criteria, SDCi exhibits very low antimicrobial activity against the tested microorganisms, indicating it is not a suitable candidate for antimicrobial use in isolation. Hence, the traditional use of C. iguanaea for treating urinary infections may be more closely associated with its notable anti-inflammatory and antinociceptive properties (Moraes et al. 2022; Almeida et al. 2024; Ribeiro et al. 2025) than with direct antimicrobial activity.

Notably, the global incidence of fungal infections continues to increase, despite the limited availability of effective antifungal agents (Tediole et al. 2025). Concurrently, ESKAPE pathogens remain leading contributors to severe healthcare-associated infections, posing a significant threat to critically ill and imm