In vitro cytotoxic and anti-inflammatory activities of Ayapana triplinervis essential oil and hydroethanolic extract

1 Introduction

Compounds in plants provide a key basis for developing synthetic or semi-synthetic analogs with improved pharmacology. These compounds aid in treating or preventing various human pathological conditions (Najmi et al., 2022). It is estimated that approximately 35% of the annual global pharmaceutical market comes from natural products or related medicines (Calixto, 2019).

Ayapana triplinervis (M.Vahl) R.M.King & H.Rob. is an aromatic herb classified into morphotype A (white japana) and morphotype B (purple japana) (Grossi and Barreto, 2024). It is popularly known as japana, white japana, purple japana, snake herb, holy herb, and chili pepper. It is distributed throughout Asia and South America. In Brazil, it occurs in the northern region, in the states of Amapá, Amazonas, and Pará (Grossi and Barreto, 2024; Nery et al., 2014).

A. triplinervis roots, stems, and leaves are used in folk medicine as antibacterial, antipyretic, anti-inflammatory and anti-melanogenic (Arung et al., 2012; Santos et al., 2023). Furthermore, compounds with potential pharmacological activities have already been identified in the oils and extracts of the species, such as terpenes, sterols, flavonoids, coumarins and others (Mala et al., 1999; Trang et al., 1993).

In addition to traditional uses, pharmacological activities have been proven by several scientific studies. The A. triplinervis leaf hydromethanolic extract showed gastroprotective action against gastric ulcers, in addition to reducing inflammatory markers, improving the redox status and showing no toxicity (Maity et al., 2021). The essential oil and the main compound (thymohydroquinone dimethyl ether) from A. triplinervis showed antiviral activity in human cells (A549) and a lack of acute toxicity in zebrafish (Haddad et al., 2019).

A. triplinervis has been reported to present different pharmacological properties. However, despite this evidence, there are still few studies that systematically investigate the cytotoxic and anti-inflammatory effects of this species. So, it is important to expand scientific knowledge about the biological potential of this species. Thus, the present study aims to evaluate, through in vitro assays, the cytotoxicity and anti-inflammatory activity of A. triplinervis essential oil and extract.

2 Materials and methods

2.1 Plant material

A. triplinervis leaves were collected in Mirasselvas, Capanema, Pará state, Brazil (1°5′34″ S/46°59′1″W), transported in airy plastic bags and stored in a shaded and ventilated place. Botanical identification was performed by Dra. Flavia C. A. Lucas. A voucher (MFS011209) was incorporated into the “Marlene Freitas da Silva” Herbarium of the Universidade do Estado do Pará, in the city of Belém, Pará, Brazil. The access to genetic heritage was registered in SISGEN under the number A090EB2.

2.2 Essential oil and hydroethanolic extraction

The leaves were placed on trays to dry at room temperature for four days. After drying, the leaves were ground. A. triplinervis essential oil (AtEO) was extracted by hydrodistillation with a modified Clevenger extractor for 3 h. The EO was centrifuged for 5 min and dehydrated with anhydrous sodium sulfate (Na₂SO₄). The total oil yield was expressed as mL/100 g of dry matter (de Lima et al., 2023). To obtain the A. triplinervis hydroethanolic extract (AtHE), the crushed leaves were extracted with a hydroethanolic solution (70% ethanol) exhaustively. Subsequently, the AtHE was concentrated in a rotary evaporator (QUIMIS Q344M2, Quimis, Brazil), followed by drying in a freeze dryer (MD-10N/C Freezer Dryer, Midea biomedical, China). Then the AtHE was stored in an amber glass ampoule and kept under refrigeration at 4 °C–8 °C, to later be used for chemical and pharmacological assays.

2.3 GC and GC-MS analyses

The EO chemical composition was analyzed using a gas chromatography coupled to mass spectrometry Shimadzu QP2010 ultra system equipped with an AOC-20i auto-injector. An Rtx-5MS silica capillary column (30 m × 0.25 mm; 0.25 μm film thickness) was used, under the following operating conditions: temperature program: 60 °C–240 °C (3 °C/min); injector temperature: 250 °C; carrier gas: helium (1 mL/min); injection of 1 µL of essential oil solution (5:500 μL of hexane) in split mode (1:20 split ratio); mass spectra were obtained by electron ionization at 70 eV; ion source temperature: 200 °C, with automatic scanning every 0.3 s, with mass fragments in the range of 35–400 m/z.

Gas chromatography-flame ionization detector (GC-FID) analysis was performed on a Shimadzu QP-2050 instrument (Shimadzu, Tokyo, Japan) equipped with an FID detector under the same conditions described above, except that hydrogen was used as the carrier gas. The percentage composition of the oil sample was calculated from the GC-FID peak areas.

The retention index was calculated for all detected components using a homologous series of C₈-C₄₀ n-alkanes (Sigma-Aldrich, Milwaukee, WI, USA), according to the linear equation of van den Dool and Kratz (van Den Dool and Kratz, 1963). The identification of chemical components was based on the linear retention index and the fragmentation pattern observed in the mass spectra, compared with authentic samples in the Adams (Adams, 2007) and FFNSC-2 (Mondello, 2011) libraries.

2.4 UHPLC-ESI-MS/MS

An AtHE cleaning step was carried out by solid phase extraction using Phenomenex Strata C18 cartridges (500 mg), previously activated with MeOH (5 mL) and equilibrated with MeOH:H₂O (5 mL, 1:1, v/v). Purified extracts were eluted from cartridges using MeOH:H₂O (5 mL, 1:1, v/v), filtered through a 0.22 μm Polytetrafluoroethylene (PTFE) filter, dried, and diluted to 10 μg/mL with HPLC solvent.

The analysis of the chemical characterization of the AtHE was carried out using Ultra High Performance Liquid Chromatography (UHPLC) Vanquish Flex (Thermo Fisher Scientific, Bremen, Germany), with an automatic sampler, binary pump. The system was coupled to a mass spectrometer (Mass Spectrometry - MS) Orbitrap Exploris™ 240 (Thermo Fisher Scientific, Bremen, Germany), with a heated electrospray ionization source (Heated Electrospray Ionization - HESI) and an Orbitrap type analyzer, operating in the range of mass/charge ratio (m/z) from 100 to 1,200, in positive and negative ionization modes, with resolution of 120,000 for full scan and 15,000 for tandem mass spectrometry (MS²). Chromatographic separations were performed on a Hypersil GOLD column (C18; 50 × 3 mm, 1.969 μm; Thermo Fisher Scientific, Runcorn, United States) operating at a column temperature of 40 °C. Four solvents were used, for the positive mode solvent (A1) 0.1% formic acid in water and (B3) 0.1% formic acid in MeOH and for the negative mode solvent (A2) 5 mM ammonium formate in water and (B2) 5 mM ammonium formate in MeOH, both at a flow rate of 0.3 mL/min. The elution gradient was defined as follows for both modes: 0–5.0 min, 10%–50% B; 5.0–9.0 min, 100% B; 9.0–12.5 min, 100% B; 12.5–12.6 min, 10% B; 12.6–15.0 min, 10% B. Injection volume was 2 μL.

The ionization source conditions were as follows: Ion spray voltage, 3.5 kV; Sheath gas flow rate, 40 (arbitrary units); auxiliary gas flow rate (Auxiliar gas), 8 (arbitrary units); Sweep gas, 1 (arbitrary units); Ion transfer tube temperature, 325 °C; vaporizer temperature, 350 °C; S Lens RF (RF Lens), 70%. Data-dependent acquisition (DDA) mode was used, based on the 5 most intense precursor ions; the isolation window was 1.5 m/z; the collision energy type has been “normalized” with HCD collision energy values of 30, 50% and 70%. For MS2 data acquisition, the following filters were applied: minimum intensity (5,000) and dynamic exclusion (exclusion after 3 acquisitions, 5 s interval, 5 ppm tolerance and isotope exclusion).

Mass spectrometry data files obtained from the Orbitrap UHPLC instrument for the extract were converted to mzXML format using MSConvert software (ProteoWizard, Palo Alto, CA, USA) (Chambers et al., 2012). Data processing was performed with MZmine version 4.0 (Schmid et al., 2023). Precursor and fragment intensity thresholds were set at 1.0 × 10³ (MS1) and 8.0 × 10¹ (MS2), respectively. Total ion chromatograms (TICs) were generated using the ADAP algorithm. Retention times were within a range of 0.02–10.00 min, with a minimum group size of 3 and an intensity threshold of 1.0 × 10³. The maximum and minimum intensity values were set at 3.0 × 10⁴, and the scanner accuracy was set at 0.002 m/z or 10 ppm. Chromatogram deconvolution was performed using the Local Minimum Resolver algorithm, applying a chromatographic threshold of 80%. The minimum search interval for retention/mobility time was 0.050, with a precursor tolerance of 0.002 m/z or 10 ppm. The noise threshold settings were 15, while the absolute minimum height was 1.0 × 10⁴. The peak-to-peak ratio was set to 1.7, the peak duration ranged from 0.10 to 1.00, and the wavelength range (RT) was from 0.05 to 0.15. Isotope detection was based on a peak window tolerance of 0.002 m/z or 10 ppm, a retention time tolerance of 0.2 min, and a maximum charge of 2. Peak alignment was conducted with an m/z tolerance of 3:1 by retention time and a retention time tolerance of 0.2 min. After alignment, the resulting data were filtered to eliminate duplicates and entries without an associated MS2 spectrum. The processed files were then exported in mgf and CSV formats for further analysis on the GNPS2 platform (Wang et al., 2016), see the metadata in positive mode at https://gnps2.org/status?task=1d01d05566b0490182f7d4978f3df698, and the negative mode at https://gnps2.org/status?task=e607f4dcf86b420ebe7baf87ac6b9531.

2.5 Cell viability assay and cytotoxic analysis (MTT)

Cell viability and cytotoxicity analysis were determined by converting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into formazan crystals, as previously described by Mosman (Mosmann, 1983).

In the cell viability test, cells (cells/mL: 1 × 10⁴ cells/well) were treated with different concentrations (500, 250, 125, 62.5, 31.25, 15.62, and 7.81 μg/mL) of AtEO, previously diluted in DMSO, and incubated for 24 h. DMSO (0.01%) was used as a negative control. The reading was performed on a plate spectrophotometer (Pollaris®, Celer, Minas Gerais) at a wavelength of 570 nm (Figueiredo et al., 2019). The percentage of cell viability was calculated from the absorbances using the equation below. Whereas, Aa (Sample absorbance) and MAc (mean absorbance of the control).

% of cell viability = Aa * 100 MAc

The cytotoxic activity of the oil was evaluated against the following tumor cell lines: gastric ascites (AGP01), cutaneous melanoma (Sk-MEL-19), glioblastoma (AHOL), lung cancer (A549), normal fibroblasts (MRC-5), and non-malignant murine macrophages (RAW 264.7). All cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, at 37 °C with 5% CO₂ atmosphere. For this purpose, the cells were treated with AtEO (0.4–25 μg/mL), previously diluted in DMSO, and incubated for 72 h. The plates were read on a plate spectrophotometer (Pollaris®, Celer, Minas Gerais) at a wavelength of 570 nm (Jaganathan et al., 2011).

The mean inhibitory concentration (IC₅₀) and respective confidence interval (CI₉₅) for each sample were obtained from nonlinear regression in the GraphPad Prism version 8.0 program. A sample’s Selectivity Index (SI) reflects its ability to target specific cells, which was determined by the following equation.

SI = IC 50 ls IC 50 lt

Whereas SI (Selectivity Index), IC (Inhibitory Concentration), ls (healthy lineage), and lt (tumor lineage) are used.

2.6 In vitro anti-inflammatory assay

Three concentrations of the same without impact on cell viability were selected from the results of cell viability in the MTT assay to evaluate the anti-inflammatory activity of AtEO and AtHE. The macrophage cell line RAW 264.7 was cultured (1 × 10⁵ cells/well) in 96-well plates, stimulated with Escherichia coli, LPS, and then treated or not (LPS group) with the oil concentrations and incubated for 24 h at 37 °C, 5% CO₂. Dexamethasone (10 µM) was used as a positive control, and DMSO 0.01% as a negative control.

2.7 Nitrite dosage

Nitrite levels were measured spectrophotometrically by forming an azo dye resulting from the reaction of nitrites in the sample with a Griess solution (Green et al., 1982). The samples were incubated with modified Griess reagent (Sigma-Aldrich®) in 96-well plates, at room temperature and in the dark for 10 min, and subsequently analyzed in a microplate reader (Foccus). A nitrite standard curve (0–100 µM) was used for quantification.

2.8 Statistical analysis

Data were presented as mean ± standard deviation (SD), adopting p < 0.05 as a statistically significant value. Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA). Comparisons between groups were made using one-way analysis of variance (ANOVA) followed by Tukey’s post-test for multiple comparisons. Each sample was analyzed from three experiments performed in triplicate.

3 Results and discussion

3.1 A. triplinervis essential oil chemical composition

Table 1 shows the 21 constituents identified in the AtEO, ordered in increasing order of their retention indexes (RI). These constituents represent 95.1% of the essential oil’s total content. The yield of the AtEO was 2.4%.

TABLE 1. Chemical composition of Ayapana triplinervis essential oil.

RI_(C), Calculated retention index; RI_(L), Literature retention index.

Adams library (Adams, 2007).

FNSC library (Mondello, 2011).

The main classes of constituents were sesquiterpene hydrocarbons (48.8%), followed by oxygenated monoterpenes (45.6%). The oxygenated monoterpene 2,5-dimethoxy-p-cymene (45.0%) was the major compound in this study. Other compounds, such as the sesquiterpene hydrocarbons β-selinene (19.2%) and (E)-caryophyllene (18.4%), were identified in smaller quantities. The chemical structures of these compounds are shown in Figure 1.

In agreement with the present research, 21 components were identified in A. triplinervis leaf essential oil from Tracuateua municipality, Pará (Brazil). 2,5-dimethoxy-p-cymene (63.6%), β-selinene (16.4%), and (E)-caryophyllene (9.4%) were the main constituents (Barros et al., 2024).

The major compound 2,5-dimethoxy-p-cymene, isolated from AtEO, showed potent antiviral activity against the Zika virus in human cells and the absence of toxicity in zebrafish (Haddad et al., 2019). The AtEO from Réunion Island (Indian Ocean) is chemically consistent with the growth stage, with 2,5-dimethoxy-p-cymene as the main compound. However, its chemical composition may vary according to the geographic location, evidencing the influence of the local environment on the plant’s chemical profile (Gauvin-Bialecki and Marodon, 2008). This result may contribute to the knowledge of intraspecific chemical variation and may be useful in developing pharmaceutical or cosmetic products based on this essential oil.

β-Selinene, present in species of the Asteraceae family, has demonstrated antioxidant and antimicrobial activities, in addition to contributing to the herbaceous aroma of essential oils, with its intensity influenced by cultivation conditions (Juteau et al., 2002). (E)-Caryophyllene stands out for its woody odor, cosmetic and food applications, and for enhancing antineoplastic effects and alleviating refractory cancer pain (Cruz et al., 2023). On the other hand, caryophyllene oxide is a natural compound with antitumor properties, which has the potential to overcome resistance by inhibiting efflux pumps, such as P-glycoprotein, inhibiting breast cancer resistance protein, a critical mechanism in cholangiocarcinoma, an aggressive cancer of the bile ducts (Ortiz-Rivero et al., 2024).

In this context, the monoterpene class is known for its antinociceptive, anti-inflammatory, antibacterial, and pharmacological properties. It plays an important role as a natural defense compound in plants (Baig et al., 2024; Barros et al., 2024). Their abundance, low cost, and applications in fragrances, food, and nutraceuticals highlight their commercial value (Wojtunik-Kulesza, 2022).

Moreover, monoterpenes are potential therapeutic agents to effectively and safely treat metastatic cancers, targeting specific molecular targets, such as matrix metalloproteinase 9, and modulating cancer-associated signaling pathways. These compounds proved promising candidates, combining effective antitumor properties with a safety profile at therapeutic doses (Varlı et al., 2024).

3.2 A. triplinervis hydroethanolic extract chemical composition

Table 2 shows the 26 constituents annotated in the AtHE, mainly belonging to the flavonoids and phenolic classes. Among the compounds identified (level 2), the chlorogenic acid feruloylquinic acid, esculin, chlorogenic acid, rutin, hyperoside, quercetin, tiliroside, among others, stand out (see Figure 2).

TABLE 2. Annotated compounds from the hydroethanolic extract of Ayapana triplinervis.

RT, Retention time.

Deprotonated precursor ion observed.

Main product ions used for structural confirmation in negative ionization mode.

Ion formed during ionization.

Similarity between two MS/MS spectra in the GNPS2 library.

Error between experimental mass and theoretical mass.

Spectra code compared in GNPS2.

Polyphenols such as protocatechuic acid, quercetin-hexoside, quercetin-hexoside-malonate, quercetin-hexoside-ramnoside, and caffeic acids isoferulic, feruoyl-hexoside, tricaffeoyl-glucarate, and isoferulic acid were also identified in A. triplinervis aqueous acetonic extract, followed by ayapin and ayapanin, known as specific coumarin derivatives in A. triplinervis plant (Taïlé et al., 2020).

The flavonoids’ ability to modulate cellular mechanisms related to the development of cancerous cells has been investigated. Compounds such as quercetin and kaempferol exhibit antitumor and anti-inflammatory activities, promoting apoptosis in cancerous cells and modulating important pathways, such as PI3K/Akt, NF-κB, MAPK, and p53, which are related to cell proliferation and survival (Chen et al., 2025). Moreover, flavonoids can also act in the modulation of pathways associated with the endoplasmic reticulum, a cellular structure involved in maintaining cellular homeostasis and in the response to intracellular stress, processes that directly influence the survival and death of tumor cells (Long et al., 2025).

This class of compounds also plays an important role in modulating inflammatory processes. Chronic inflammation is associated with inflammatory bowel disease and arthritis. These compounds reduce pro-inflammatory cytokines and inhibit signaling pathways such as NF-κB, AP-1, and COX-2. They also help reduce oxidative stress (Malhotra et al., 2026).

The presence of phenolic compounds may be associated with anti-inflammatory activity, since these secondary metabolites have a recognized ability to modulate inflammatory mediators and reduce inflammatory processes in in vivo experimental models (Chlif et al., 2022). Similarly, the phenolic fraction of plant extracts has been shown to have a significant inhibitory effect in the inflammatory models evaluated (Küpeli et al., 2007).

In addition to anti-inflammatory properties, phenolic compounds have also been associated with cytotoxic effects against different tumor cell lines. Thus, the cytotoxic effects contained in the extracts can be attributed to the presence of phenolic compounds, mainly rutin and chlorogenic acid, which are associated with the MDA-MB-231 breast cancer cell line (Januškevičė et al., 2025). Furthermore, phenolic compounds reduced the viability of prostate cancer cells (DU145, PC3 and LNCaP) and induced apoptosis (Maheshwari and Sharma, 2023).

3.3 Anti-inflammatory activity of Ayapana triplinervis

3.3.1 Effects of Ayapana triplivervis on cell viability

To evaluate the cell viability, AtEO and AtHE were tested on a macrophage cell line (RAW 264.7) and Normal fibroblasts (MRC-5), respectively. This allows us to define the most suitable concentrations for anti-inflammatory evaluation. The treatment with AtEO and AtHE reduced the cell viability. AtEO reduced the cell viability at all concentrations (1.56–100 μg/mL), and AtHE reduced the cell viability at the highest concentrations (12-2-100 μg/mL). Therefore, based on these results, the AtEO was more active than the AtHE, see Figure 3.

3.3.2 Anti-inflammatory potential of Ayapana triplinervis

Inflammation is a protective response to infections and vascularized injuries, triggered by microorganisms, chemical or physical stimuli, foreign objects and tissue necrosis. It is characterized by the release of cytokines and metabolites, such as TNF-α, lipoxin, prostaglandin and leukotrienes, as well as the recruitment of leukocytes, leading to the five classic signs: pain, swelling, edema, death and loss of function (Soares et al., 2023).

To evaluate the anti-inflammatory potential of A. triplinervis, the RAW 264.7 macrophage model was used; macrophages were treated or not (LPS group) with the three selected concentrations (0.195, 0.39, and 0.78 μg/mL). A significant increase in nitrite production was observed in cells stimulated with LPS compared to the negative control. After treatment with AtEO and AtHE in cells previously stimulated with LPS (Figure 4), there was a significant reduction in nitrite expression at all tested concentrations when compared to the LPS group (p < 0.05).

The AtEO displayed anti-inflammatory effects, significantly reducing ear edema in preclinical trials using mice (Barros et al., 2024). Regarding the major compound, studies have reported the anti-inflammatory activity of (E)-caryophyllene, where it was able to inhibit neutrophil migration and decrease the total and differential leukocyte count in acute inflammation and infection in a sepsis model (Brito et al., 2019). (E)-caryophyllene also showed increased levels of anti-inflammatory cytokines and downregulation of pro-inflammatory cytokines in a rat model of cutaneous wound excisions (Gushiken et al., 2022). In another study, (E)-caryophyllene was the main constituent found in the Salvia aurea essential oil, inhibiting NO production and preventing nuclear translocation of NF-κB, consequently decreasing the expression of iNOS and COX-2, two pro-inflammatory enzymes relevant to the inflammatory response (Alves-Silva et al., 2023).

The main constituent found in the AtEO, 2,5-dimethoxy-p-cymene, inhibited NO production in LPS-stimulated murine RAW 264.7 macrophage cells (Pham et al., 2023). Moreover, β-selinene demonstrated an anti-inflammatory effect in inhibiting leukocyte migration (Soares et al., 2022). So, these results suggest that the effects observed in A. triplinervis may be associated with the presence of these compounds, which may act synergistically.

Talking about the AtHE, treatment with the AtHE, rich in polyphenols, significantly reduced the expression of the NFκB gene and the secretion of IL-6, demonstrating its anti-inflammatory potential (Taïlé et al., 2020). Moreover, 7-methoxycoumarin isolated from A. triplinervis reduces the paw edema in the carrageenan-induced model mediated by COX-2, TNF-α, and IL-1β (Cheriyan et al., 2026). These findings indicate that A. triplinervis plays a significant role in the regulation of inflammatory processes, reinforcing its therapeutic relevance in inflammatory conditions.

3.4 Cytotoxic activity of the essential oil and hydroethanolic extract of Ayapana triplinervis

The antiproliferative effects of AtEO and AtHE were evaluated against human cancer cell lines AHOL1 (human glioblastoma), cutaneous melanoma (Sk-MEL-19), AGP-01 (gastric ascites), A549 (lung cancer), and against a healthy cell line, RAW 264.7 (murine macrophages), and MRC-5 (normal fibroblasts) using the MTT assay. IC₅₀ values less than 25 μg/mL and 50 μg/mL in at least 1 cell line were considered active for AtOE and AtHE, respectively (Figueiredo et al., 2019).

Based on cytotoxicity relative to a normal cell line, the selective index was calculated for each sample. IS values equal to or greater than 2 indicate differential selectivity, while an IS below 2 indicates general compound toxicity. The IC₅₀ and IS values are presented in Table 3.

TABLE 3. Cytotoxic activity of Ayapana triplinervis.

The values are expressed as IC₅₀ (median inhibitory concentration of cell growth in µg/mL); 95% confidence intervals obtained by non-linear regression for all cell samples; ≥ 2 was adopted as the value to be considered selective; IS, Selectivity Index; AtOE, A. triplinervis essential oil; AtHE, A. triplinervis hydroethanolic extract; ND, Not determined; SI, IC₅₀ in MRC-5 or RAW 264.7 cells/IC₅₀ in a cancer cell (superscript value).

AtEO and AtHE showed cytotoxic activity against all human tumor cell lines, AHOL1 (IC₅₀ 5.75 μg/mL), AGP-01 (AtEO IC₅₀ 12.53 μg/mL; AtHE: IC₅₀ 30.45 μg/mL), A549 (AtEO: IC₅₀ 6.56 μg/mL; AtHE: IC₅₀ 35.1 μg/mL), Sk-MEL-19 (AtHE: IC₅₀ 27.01 μg/mL), as well as in the healthy cell lines RAW 264.7 (AtEO: IC₅₀ 13.41 μg/mL), and MRC-5 (AtHE: IC₅₀ 29.22 μg/mL).

Although there are no reports regarding the cytotoxic activity of AtEO and AtHE in cancer cell lines, species that produce constituents found in the present study can endorse this activity. 2,5-dimethoxy-p-cymene, the main chemical component in the roots of Carpesium cernuum (54.8%), was toxic against cancer cell lines derived from cutaneous malignant melanoma, A375 (IC₅₀ 71.66 nLmL) and C32 (IC₅₀ 107.2 nL/mL) (Wajs-Bonikowska et al., 2021).

Cancer is a group of diseases that can affect any part of the body and is characterized by rapid and uncontrolled cell growth (WHO, 2026). Mechanisms of neoplastic initiation and development involve genetic mutations and epigenetic changes that cause deregulation of the normal controls of cell division, programmed cell death, and DNA repair (Saran and Thakur, 2024).

The essential oil from Pluchea indica (Asteraceae), which contained β-selinene (42.0%–43.5%) as the main compound, showed cytotoxic activity in four cancer cell lines K562 (leukemia, IC₅₀ 19.95 μg/mL), HeLa (cervical adenocarcinoma, IC₅₀ 40.94 μg/mL), HepG2 (liver carcinoma, IC₅0 34.78 μg/mL), and MCF-7 (breast adenocarcinoma, IC₅₀ 26.63 μg/mL) (Trang et al., 2024). Moreover, the leaf and stem essential oils from Blepharispermum hirtum (Asteraceae), rich in β-selinene, demonstrated inhibition against the MDA-MB-231 cell line (breast cancer, IC₅₀ 88.4 ± 0.5 and 123.6 ± 0.8 μg/mL, respectively). The authors suggest a possible selectivity for malignant cells compared to healthy cells (MCF-10A, IC₅₀ > 300 μg/mL), since tumor cells showed greater susceptibility to treatment. However, the study does not present the calculation of the selectivity index (Shah et al., 2022).

Similarly, the acetone (IC₅₀ 180.12 μg/mL), methanol (IC₅₀ 298.25 μg/mL), and ethanol (IC₅₀ 410.23 μg/mL) extracts from Xanthium strumarium (Asteraceae) inhibited the viability of K562 cells (human chronic myeloid leukemia) and the P3HR-1 cell line (human Burkitt lymphoma IC₅₀ 221.81; 380.62 and >500 μg/mL, for each extract respectively). This effect occurred through the induction of apoptosis, evidenced by the release of cytochrome c, the expression of caspase-3 and caspase-8, and increased DNA fragmentation. Taken together, these results reinforce the therapeutic potential of the species and its bioactive constituents as promising sources of molecules with anticancer action (Beyatli, 2025).

The selectivity index (SI), calculated as the ratio between the IC₅₀ of normal cells and the IC₅₀ of tumor cells (Badisa et al., 2009) indicating that the AtEO exhibited approximately twice the cytotoxic selectivity in the AHOL1 (2.75) and A549 (2.04) cell lines, compared to the macrophage cell line, see Table 3. Moreover, AtHE has limited selectivity for the Sk-MEL-19 (0.924), AGP-01 (1.04), and A549 (1.201) cell lines.

So, these findings demonstrate consistency with the results obtained in the cytotoxicity analysis of the present study, since the AtEO and AtHE showed in vitro cytotoxic activity, and this activity can be associated with the main components found.

4 Conclusion

The results demonstrated that the essential oil and hydroethanolic extract of A. triplinervis exhibited cytotoxic activity against human tumor cell lines, with moderate selectivity for the essential oil and no selectivity for the extract, in addition to the ability to modulate nitrite levels, highlighting its cytotoxic and anti-inflammatory potential in controlled culture environments. So, this work reports the chemical composition of Ayapana triplinervis hydroethanolic extract and its cytotoxic activity for the first time. Furthermore, there appears to be consistency in the chemical composition of the essential oil of this species, which is rich in 5-dimethoxy-p-cymene, and a great deal of chemical variability in the non-volatile compounds.

These findings suggest the therapeutic potential of A. triplinervis essential oil and hydroethanolic extract and indicate the need for further investigations in complementary in vitro and in vivo models for a better understanding of their effects, aiming at their application as adjuvant therapy against cancer and inflammatory conditions.

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Footnotes

Edited by: Lucia Pinheiro Santos Pimenta, Federal University of Minas Gerais, Brazil

Reviewed by: Chengqian Pan, Jiangsu University, China

Subhabrata Guha, Chittaranjan National Cancer Institute (CNCI), India