Seasonal Dynamics of the Essential Oil Constituents from the Aerial Parts of Vernonanthura polyanthes (Asteraceae) and Their Anti-Leishmania infantum Potential: A Multimethodological Approach

TL;DR

This study tracked how the chemical makeup and antileishmanial activity of Vernonanthura polyanthes essential oils changed over two years, identifying key compounds linked to effectiveness.

Contribution

The study reveals seasonal biomarkers in Vernonanthura polyanthes essential oils that correlate with antileishmanial activity and low cytotoxicity.

Findings

Hydrocarbon sesquiterpenes like β-caryophyllene and β-pinene are strongly correlated with antileishmanial activity.

Oxygenated sesquiterpenes in late 2024 reduced potency despite high concentrations.

Multivariate analysis explained 61% of the variance in activity, linking chemical profiles to biological effects.

Abstract

The present study investigated the chemical composition and antileishmanial activity of Vernonanthura polyanthes essential oils over a two-year monitoring period (monthly collection from January/2023 to December/2024). The oils exhibited a high concentration of hydrocarbon sesquiterpenes, primarily germacrene D, β-caryophyllene, α-humulene, and bicyclogermacrene, whereas the levels of monoterpenes and oxygenated sesquiterpenes fluctuated seasonally. Activity against promastigotes of Leishmania (L.) infantum was strongly dependent on the essential oil chemical profile, with consistently low EC50 values seen in months with a higher content of hydrocarbon sesquiterpenes. However, significant increases in oxygenated sesquiterpenes at the end of 2024 were accompanied by a reduction in potency. Cytotoxicity against NCTC cells remained low in most samples (CC50 > 200 µg/mL). Multivariate statistical analysis was applied to investigate the relationship between the chemical composition of studied essential oils and their antileishmanial activity. Partial Least Squares (PLS) modeling based on key volatile markers (VIP > 1.0) revealed a significant correlation between constituent profiles and biological potency, explaining 61% of the activity variance (R2 = 0.61). Regression coefficients identified β-caryophyllene and β-pinene as major contributors to enhanced activity, while β-bourbonene was associated with reduced potency. Seasonal evaluation showed that β-caryophyllene provides stable baseline activity, whereas peaks in β-pinene correspond to increased potency, suggesting a positive correlation associated with enhanced potency between these constituents. Integrated EC50 and CC50 models further demonstrated that β-pinene and β-caryophyllene combine desirable features of strong activity and low cytotoxicity. These findings clarify the metabolic drivers of seasonal bioactivity in V. polyanthes and highlight key biomarkers that may guide future pharmacological and biotechnological applications.

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1. Introduction

Visceral leishmaniasis (VL) remains one of the most severely neglected tropical diseases worldwide. According to the World Health Organization (WHO), an estimated 50,000 to 90,000 new VL cases occur annually, although only 25–45% are actually reported [1]. The WHO also highlights that more than one billion people live in endemic areas and are at risk of infection. In the Americas, the Pan American Health Organization (PAHO) reports that approximately 96% of VL notifications occur in Brazil, underscoring the substantial national burden of the disease [2]. Current treatment for visceral leishmaniasis relies on pentavalent antimonials, amphotericin B (deoxycholate or liposomal formulations), and, in specific contexts, miltefosine. However, these therapies present important limitations since antimonials and miltefosine are highly toxic and require prolonged regimens. On the other hand, amphotericin B, although more effective, is associated with significant adverse effects and requires inpatient administration [3,4]. In this scenario, efforts to develop more effective and less harmful therapeutic alternatives are essential and natural products are an important source of new bioactive compounds [5].

The genus Vernonanthura (Asteraceae), formerly included in Vernonia, comprises species widely distributed in South America, especially in Brazil, where they are used in traditional medicine for the treatment of inflammations, infections, and gastrointestinal disorders [6]. Despite being relatively under-researched from a phytochemical standpoint, the genus presents a characteristic set of secondary metabolites, notably sesquiterpenes, both volatile and non-volatile [7]. The essential oils of Vernonanthura are typically rich in sesquiterpene hydrocarbons, such as germacrene D, β-caryophyllene, and α-humulene, in addition to oxygenated derivatives in smaller proportions [8]. This volatile pattern, generally poor in monoterpenes, constitutes a relevant chemical marker of the genus. Among the non-volatile metabolites, sesquiterpene lactones of the germacranolide, heliangolide, and guaianolide types stand out, recognized for their anti-inflammatory, cytotoxic, and antiparasitic activities [9]. Flavonoids, phenolic acids, and triterpenes also occur, contributing to the chemical diversity and pharmacological potential of the species [10]. In general, the predominance of sesquiterpenes and the presence of phenolic compounds support both the biological interest and the ethnobotanical value of Vernonanthura, justifying further investigations aimed at chemical characterization and evaluation of its bioactive properties.

Despite of Vernonanthura polyanthes (commonly known in Brazil as “assa-peixe”) have had their essential oils previously investigated [11,12], no studies have addressed the seasonal variation in its volatile composition or how such compositional changes may influence biological activity. At this point, our research group reported the antiparasitic effects of essential oils of different Brazilian plant species [13,14]. As part of this continuous study, the essential oils from aerial parts of V. polyanthes extracted monthly from January 2023 to December 2024 were chemically analyzed and were tested in vitro against promastigote forms of L. infantum. In order to measure the importance of the identified constituents of each studied oil for the antileishmanial activity, was employed computational approaches such as Partial Least Squares Regression (PLSR). The validation and interpretation of the results led to the selection of main attributes for an effective antileishmanial property of essential oils from aerial parts of V. polyanthes.

2. Results and Discussion

2.1. Chemical Composition of the Essential Oils from Aerial Parts of V. polyanthes

The essential oils from aerial parts of V. polyanthes collected monthly from a single specimen from January 2023 to December 2024 were individually obtained by hydrodistillation using a Clevenger apparatus. The identification of the individual compounds of each oil was achieved by interpretation of mass spectra recorded using a tandem gas chromatography/mass spectrometry (GC/MS) based on those reported in the literature [15] and also by calculation of their respective arithmetic index (AI), determined relative to the retention times of a series of n-alkanes (see Material and Methods—Molecular Dereplication) Supplementary Materials. Obtained results, as indicated on Table 1 and Table 2, revealed a highly consistent profile, characterized by the predominance of sesquiterpenes, mainly hydrocarbons.

The proportion of sesquiterpene hydrocarbons varied between 93.4–98.8% in 2023 and 82.0–96.7% in 2024, demonstrating relative interannual stability, although with a more pronounced reduction in the final months of 2024. In contrast, monoterpenes occurred only in small proportions (0–3.3%), with slight increases during warmer months, while oxygenated sesquiterpenes remained at low levels for most of the period, except for a marked increase observed between October and December 2024 (up to 15.8%).

Among the major constituents, germacrene D, β-caryophyllene, α-humulene, and bicyclogermacrene were detected in all months, composing the structural core of the essential oil. Germacrene D showed variations of 17.2–26.6% in 2023 and 18.9–28.4% in 2024, while bicyclogermacrene ranged from 15.2–23.2% in 2023 to 16.8–23.8% in 2024. In parallel, β-caryophyllene and α-humulene oscillated between 14.7–21.3% and 14.8–23.1% in 2023, respectively, and 13.7–19.5% and 13.1–19.4% in 2024. Considering that these compounds were predominant in the studied oils, a germacrene D/β-caryophyllene/α-humulene chemotype was suggested. Previous studies on the essential oil of V. polyanthes reported zerumbone (15.8%), bicyclogermacrene (8.9%), α-humulene (4.8%), and germacrene D (4.3%) as the major constituents [11,12]. With the exception of zerumbone, which was not detected in the analyzed oils, a similar chemical profile was observed compared to the previously reported study. In contrast, myrcene was previously described as the predominant monoterpene (34.3%). However, in the present study, myrcene was not detected, with monoterpenes occurring only in minor amounts, the highest being observed at June 2024 (3.2%). Although the qualitative composition remained predominantly stable, relevant differences were observed at the end of 2024, when the relative proportion of oxygenated compounds, such as spathulenol, viridiflorol, ledol, τ-muurolol, and α-cadinol, increased, suggesting a possible intensification of oxidation processes or physiological changes associated with more severe environmental conditions. In November and December 2024, this class reached its highest values (14.4–15.8%), concomitantly with a reduction in the fraction of sesquiterpene hydrocarbons.

The essential oil yield ranged from 0.013–0.031% in 2023 and from 0.010–0.022% in 2024, with slightly lower values observed in the latter year. This variation suggests that environmental factors may influence the biosynthesis and accumulation of volatile compounds, including seasonality, circadian rhythm, developmental stage and age, temperature, water availability, ultraviolet radiation (UV), soil nutrients, altitude, atmospheric composition, and tissue damage [16]. However, because several of these parameters were not recorded during the collection periods, it was not possible to draw more robust conclusions regarding their effects on the chemical composition of the oils studied. However, the results obtained indicate that V. polyanthes showed a stable chemical profile, but with seasonal and interannual fluctuations that reflect physiological adaptations of the species to varying climatic conditions [17].

2.2. Evaluation of Antileishmanial Activity of Essential Oils from V. polyanthes

Based on previous evidence indicating that essential oils exhibit in vitro anti-leishmanial activity [18,19], the essential oils from aerial parts of V. polyanthes were evaluated against promastigote forms of L. infantum. Their cytotoxicity toward NCTC cells was also assessed, as shown in Table 3.

The biological activity of the evaluated essential oils showed clear variations over the two years of monitoring, and these oscillations showed a strong relationship with seasonal and interannual changes in chemical composition. In 2023, the essential oil was characterized by a marked predominance of hydrocarbon sesquiterpenes, frequently above 96%, including germacrene D, β-caryophyllene, α-humulene, and bicyclogermacrene, while monoterpenes remained in low proportions and oxygenated sesquiterpenes rarely exceeded 3%. This composition, relatively stable throughout the year, coincides with consistently low 50% effective concentration values (EC_50_ = 5.5–6.9 µg/mL), suggesting that the major sesquiterpenes are primarily responsible for the observed activity. Previous studies reported that the main sesquiterpenes found in the V. polyanthes essential oils, including germacrene D, β-caryophyllene, and α-humulene displayed activity against Leishmania ssp., which is consistent with the patterns found [20,21,22]. The correlation between months with higher levels of these compounds and lower EC_50_ reinforces the importance of this chemical group in the activity of the essential oil studied. Conversely, months with small proportional reductions in these constituents—such as July 2023—showed a relative loss of potency, indicating that even subtle variations in the proportion of these sesquiterpenes can impact biological activity.

In 2024, however, greater variability was observed in the chemical profile, especially from August onwards and with greater intensity at the end of the year, when the levels of oxygenated sesquiterpenes increased substantially, reaching 15.8% in November and 14.4% in December. At the same time, the fraction of sesquiterpene hydrocarbons underwent a significant reduction, reaching the lowest value recorded in the study (82% in November and December). This transition was also accompanied by changes in biological activity. Although a moderate increase in oxygenated compounds may favor activity, as observed in October 2024, when oxygenated compounds totaled 3.15% and EC_50_ reached one of the lowest values in the study (4.7 µg/mL), excessive levels seem to have the opposite effect. In November and December 2024, concomitantly with the accumulation of these compounds, there was a reduction in potency (EC_50_ = 7.0–10.3 µg/mL), indicating that the increase in oxygenated sesquiterpenes above a certain threshold may dilute the most active constituents or generate metabolites with lower efficacy. This biphasic behavior is consistent with studies that demonstrate that certain oxygenated sesquiterpene derivatives can exhibit moderate antiparasitic activity. However, at the same time, their presence in very high proportions can negatively interfere with synergistic interactions between the sesquiterpene hydrocarbons that characterize the active chemotype, as previously observed in the literature on the essential oils of Guarea macrophylla [23]. Thus, the results suggest that maintaining a balance between hydrocarbons (predominant) and oxygenated compounds (present at moderate levels) appears to be crucial for achieving greater biological efficacy of the oil.

Cytotoxicity, on the other hand, remained low for most of the studied oils (50% cytotoxic concentration—CC_50_ > 200 µg/mL), indicating good safety and reinforcing that changes in EC_50_ are predominantly due to chemical alterations, and not to increased cellular toxicity. The months with the best activity/toxicity ratios—particularly March 2024, April 2024, February 2023, and October 2024 —coincide with chemical profiles dominated by sesquiterpene hydrocarbons, reinforcing the role of this group as a chemobiological marker of the species [8].

Collectively, the data show that the biological activity of essential oils is highly dependent on chemical composition, especially the ratio between sesquiterpene hydrocarbons and oxygenated compounds. The predominance of the former is associated with high potency and toxicity, while sharp increases in the latter tend to reduce efficacy. These results highlight not only the influence of seasonal and environmental factors on the metabolic profile of the species, but also the need for continuous monitoring of volatile constituents in pharmacological studies, aiming at selecting more suitable periods for collection and subsequent biotechnological application.

2.3. Model Performance and Statistical Validation

The optimized partial least-squares (PLS) analysis, based on the selected chemical markers (Variable Importance in Projection—VIP > 1.0), revealed a significant linear correlation between the essential oil composition and biological potency. The model explained approximately 61% of the total variance in activity (R^2^ = 0.61), a robust value considering the intrinsic variability of complex biological assays and the influence of uncontrolled environmental factors. Statistical validity was confirmed by the permutation test (n = 100), demonstrating that the quality metrics of the experimental model significantly exceeded those obtained from random permutations (p < 0.05).

2.4. Identification of Potency Biomarkers

Figure 1 shows the regression coefficient of each compound present in the extract. We can observe that some constituents display negative coefficients, whereas others show positive values. For an extract to exhibit significant biological activity (lower EC_50_ values), a combined effect is expected: constituents with positive coefficients should occur at lower concentrations (e.g., β-bourbonene), while those with negative coefficients (e.g., the sesquiterpene β-caryophyllene and the monoterpene β-pinene) should be present at higher concentrations for the extract to display enhanced biological activity.

2.5. Seasonal Dynamics and Cooperative Effect

The temporal assessment of the markers (Figure 2) revealed distinct patterns of metabolic production. β-Caryophyllene showed a relatively constant proportion throughout the monitored period. This consistency suggests that it may be responsible for the baseline activity of the essential oil, ensuring a minimum level of efficacy regardless of the season.

In contrast, β-pinene exhibited pronounced seasonal fluctuations. Notably, periods of peak concentration of this monoterpene coincided with the lowest EC_50_ values (indicating greater potency), whereas its absence from the chemical profile was associated with a decline in biological activity. These findings indicate a statistical association, suggesting that the presence of β-pinene is correlated with enhanced activity, suggesting that the baseline activity provided by β-caryophyllene is enhanced in the presence of β-pinene, and that the biological activity of the extract may be compromised when it is lacking. Conversely, periods of reduced activity often coincided with production peaks of the negative marker, β-bourbonene.

2.6. Integrated Profile of EC50 and CC50 Coefficients

To assess the therapeutic potential of the biomarkers, an integrated analysis of the regression coefficients for the models related to biological activity (EC_50_) and cytotoxicity (CC_50_) was performed, as shown in Figure 3. The plot reveals that β-pinene and β-caryophyllene exhibit the ideal biological profile (opposing bars): both display negative coefficients in the EC_50_ regression model and positive coefficients in the CC_50_ regression model.

The use of Multivariate Statistical Analysis (MSA) is particularly powerful for interpreting complex datasets, as it enables the identification of the individual contribution of each constituent within a holistic framework. In this study, the application of Partial Least Squares Regression (PLSR) and Variable Importance in Projection (VIP) analyses produced consistent and complementary results, strengthening the interpretation of the antileishmanial activity. This approach allowed us to distinguish and accurately characterize the influence of each constituent of the essential oils from V. polyanthes aerial parts against promastigote forms of L. infantum, based on a systematic metabolomics-guided evaluation. Further studies with isolated compounds are needed to confirm any synergistic mechanisms.

3. Materials and Methods

3.1. Plant Material

The V. polyanthes specimen was located on the campus of the Federal University of ABC (UFABC), in Santo André, São Paulo, Brazil (23°38′35.8″ S, 46°31′44.4″ W), and compared with the voucher specimen Antar-131 deposited in the Herbarium of the Institute of Botany of the University of São Paulo (IB-USP), SisGen A483B45.

3.2. Essential Oil Extraction

From January/2023 to December/2024 (monthly collection from a single specimen), approximately 400 g of fresh aerial parts was subjected to hydrodistillation using a Clevenger-type apparatus for 5 h. After distillation, the obtained oils were extracted with ethyl ether (4 × 2 mL) and was dried over anhydrous Na_2_SO_4_. After filtration and air-drying to eliminate residual solvent, the samples were transferred to 1.5 mL borosilicate vials (11.6 × 32 mm), sealed with a manual hand crimper, and stored at −25 °C until further analysis.

3.3. Molecular Dereplication

Each obtained essential oil was analyzed, in triplicate (three injections of the same sample), using a Shimadzu GC-2010 gas chromatograph equipped with a flame ionization detector (FID), a RtX-5 capillary column (5% phenyl, 95% polydimethylsiloxane, 30 m × 0.25 mm × 0.25 μm film thickness, Restek, Anaheim, CA, USA), and an automatic injector (Shimadzu AOC-20i, Tokyo, Japan). To perform the chromatographic analysis, 1.0 μL of each sample at 1 mg/mL in ethyl ether was injected at 225 °C with helium as the carrier gas at a flow rate of 0.73 mL/min. Chromatographic method: 60 °C (2 min), 60–240 °C at 3 °C/min, then isothermal at 240 °C (10 min). Gas chromatography-mass spectrometry (GC–MS) analysis was performed on a Shimadzu^®^ GCMS-QP2010 Plus system using a DB-5MS column (5% phenyl, 95% polydimethylsiloxane, 30 m × 0.25 mm; 0.25 μm film thickness) at the same conditions described above to FID-GC analysis. The mass detection (electron ionization detector at 70 eV) was carried out at m/z range of 40–500 Da. The constituents were thus identified by comparing the obtained mass spectra with those available in the Wiley (spectrabase.com) and NIST (webbook.nist.gov/chemistry) spectral libraries, as well as by comparing the respective arithmetic indices (AI) with previously reported values [15]. For AI calculation, a homologous series of n-alkanes (C_8_–C_20_) was injected under the same analytical conditions, and the values were determined using the Van den Dool and Kratz equation [24].

3.4. Determination of Antileishmanial Activity

Isolated promastigotes of Leishmania (L.) infantum (MHOM/BR/1972/LD) were maintained in M-199 medium supplemented with 10% calf serum and 0.25% hemin at 24 °C. Promastigotes were counted in a Neubauer hemocytometer and seeded at 1 × 10^6^/well to obtain a final volume of 150 μL. To determine the antileishmanial potential, essential oils and positive control miltefosine (100% of purity—Sigma-Aldrich, St. Louis, MO, USA) were tested at top concentration 200 μg/mL and were 2-fold serially diluted into seven concentrations (100, 50, 25, 12.5, 6.75, 3.37, and 1.69 μg/mL) in dimethyl sulfoxide (concentration < 0.5% v/v). Each point was tested in duplicate. The plate was incubated for 48 h at 25 °C and the viability of promastigotes was verified by morphology in light microscopy and by the MTT assay. Briefly, MTT (5 μg/mL) was dissolved in PBS, sterilized through 0.22 μm membranes and added (20 μL/well) for 4 h at 24 °C. Promastigotes were incubated without compounds and used as viability control. Formazan extraction was performed using 10% SDS for 18 h (80 μL/well) at 24 °C and the optical density (OD) was determined in a Multiskan MS (UNISCIENCE, Miami Lakes, FL, USA) at 570 nm. One hundred percent (100%) viability was expressed based on the OD of control promastigotes after normalization [25,26].

3.5. Determination of Cytotoxicity

The essential oils and miltefosine were dissolved in dimethyl sulfoxide (concentration < 0.5% v/v) and diluted in 96-well plates using RPMI-1640 medium to final concentrations ranging from 200 to 1.69 µg/mL. The plates were then incubated with mouse fibroblast cells NCTC clone 929 cells (ATCC^®^, Manassas, VA, USA, CCL-1™ RRID:CVCL_0462) at 6 × 10^4^ cells/well for 48 h in an incubator at 37 °C with 5% CO_2_. Cell viability was determined by assessing mitochondrial metabolic activity using the MTT assay [26,27], followed by spectrophotometric reading at 570 nm.

3.6. Statistical Analysis

The CC_50_ and EC_50_ values were determined using dose–response sigmoid curves. Statistical significance between samples was assessed using p-values obtained through one-way ANOVA followed by Tukey’s multiple comparison test. All analyses were performed using GraphPad Prism 5 software. The samples were tested in duplicate, and each experiment was repeated at least twice.

3.7. Multivariate Statistical Analysis

To investigate the correlation between the seasonal volatile chemical profile (X-matrix) and the experimental biological activity (EC_50_, Y-vector) against L. infantum promastigotes, the Partial Least Squares Regression (PLSR) technique was employed [28]. Prior to modeling, the data were pre-processed to remove instrumental noise (exclusion of variables with more than 50% missing values) and scaled using unit variance (UV) [29]. This method standardizes the data by dividing each variable by its standard deviation, ensuring that all compounds (regardless of their relative abundance) have the same variance (of one) and thus a comparable importance to influence the model construction. To obtain a chemically interpretable model, a variable selection step based on the Variable Importance in Projection (VIP) criterion was applied [30], retaining only constituents with VIP scores greater than 1.0 in the final model. Model quality was assessed using the coefficient of determination (R^2^), and statistical significance was validated through a permutation test (n = 100) [31], by evaluating the intercept of the regression line from the permuted models to rule out overfitting. Additionally, a secondary model was constructed using cytotoxicity data (CC_50_) to assess the safety profile of the identified markers.

4. Conclusions

This study demonstrates that the essential oils of Vernonanthura polyanthes exhibit a chemical profile markedly dominated by sesquiterpene hydrocarbons, with variable contributions of monoterpenes and oxygenated sesquiterpenes throughout the analyzed period. The chemical composition was directly associated with biological activity and cytotoxicity, highlighting the close relationship between seasonal fluctuations, environmental variations, and the bioactive potential of the samples. The results reveal that months with a higher proportion of germacrene D, bicyclogermacrene, β-caryophyllene, α-humulene, and other sesquiterpene hydrocarbons exhibited the best EC_50_ values, reinforcing the role of these compounds as functional markers of the active chemotype of the species. On the other hand, significant increases in oxygenated sesquiterpenes, especially at the end of 2024, were associated with a reduction in biological potency, suggesting that an excess of oxidized derivatives compromises the activity of the essential oil by altering the balance between active and less effective compounds. Cytotoxicity remained relatively low throughout the study, indicating a good safety margin and reinforcing that the variations observed in activity and cytotoxicity are due to chemical changes, and not intrinsic toxicity. Taken together, the findings demonstrate that V. polyanthes has high biotechnological and pharmacological potential, but reinforce the importance of considering seasonal and environmental factors for the standardization of biological activity. Identifying periods of peak potency and toxicity contributes to optimizing collection and processing strategies for the species, as well as guiding future studies focused on isolating bioactive substances, understanding mechanisms of action, and applying them in therapeutic models.