Chemical Composition and In Vitro and In Silico Biological Activities of Myrciaria tenella (DC.) O.Berg (Myrtaceae) Essential Oil from Brazil

Myrciariatenella O.Berg, a native plant species of Brazil, exhibits pharmacological applications, including antitumor activity. In this study, we isolated the essential oil (EO) of M. tenella and identified its phytochemical profile. In addition, we determined the in vitro and in silico cytotoxic activities of EO in nontumor and tumor cell lines (gingival fibroblasts and oral squamous cell carcinoma, respectively) and its free radical scavenging activity (i.e., antioxidant activity) using ABTS and DPPH assays. The EO of M. tenella primarily constitutes hydrocarbons and oxygenated sesquiterpenes, with (E)-caryophyllene (33.95%), δ-cadinene (7.4%), caryophyllene oxide (4.74%), and viridiflorene (4.49%) as its four major components. EO effectively suppressed the cell viability of CAL-27 tumor cells to below 70% at concentrations of 125 and 250 μg/mL and exhibited a free radical inhibition potential of 75.63 ± 0.41% and 28.46 ± 0.36%, respectively, in the DPPH and ABTS assays. This chemical and biological potential may be attributed to the major compounds present in EO, as well as the molecular coupling simulations conducted, which revealed the anticancer mechanism of EO in the sesquiterpenes (E)-caryophyllene, δ-cadinene, caryophyllene oxide, and viridiflorene.


Introduction
Aromatic plants have been used in ancient civilizations to treat various diseases [1].To date, researchers have extracted various pharmacologically active molecules from plant species, which are cost-efective, less toxic, and more accessible compared with synthetic drugs [2,3].Te volatile compounds present in essential oils (EOs) are biosynthesized as secondary metabolites of aromatic plants.Tese are complex, lipophilic, and volatile mixtures that can be extracted from buds, fruits, fowers, roots, leaves, and branches.Empirical data on EOs reveal their pharmaceutical potential, emphasizing the need to explore their bioactivities [4].
Te genus Myrciaria of the plant family Myrtaceae comprises approximately 30 species that are distributed predominantly among countries on the American continent [5].Twenty-two species of Myrciaria, including Myrciaria tenella O.Berg, are native to Brazil.Te leaves of M. tenella, known as cambuí-açu or jabuticaba-macia, exhibit anti-infammatory, antibacterial, and astringent properties and are widely used by traditional communities [6,7].In vivo and in vitro studies have shown that M. tenella metabolites reduce edema, capture free radicals that trigger oxidative stress, and exhibit antimicrobial activities.M. tenella, originating from the Amazon, exhibits antioxidant potential by suppressing the overproduction of reactive oxygen species, which trigger diseases and degenerative processes [8].Among these processes, the triggering of cancer, which is associated with the excess of free radicals in the body, is noteworthy [9].Terefore, it is feasible to explore new alternatives, particularly in natural products, aligning with studies on M. tenella that have reported its antiproliferative activity against cancer cell lines [10][11][12].
In vitro tests enable researchers to analyze the cellular behavior of drugs in a controlled environment, which can help minimize their side efects [13].According to the ISO 10993-5 guidelines published in 2009, in vitro cytotoxicity tests must expose cells directly or indirectly to the desired substance [14,15].Such exposure is crucial to evaluate drug biocompatibility and cell viability, including essential cellular functions, such as growth, reproduction, and metabolism [16].
Te cytotoxic activity of the EO of M. tenella has not been investigated in cells obtained from the human oral cavity thus far.Terefore, we conducted in vitro studies on the EO of M. tenella to determine its antioxidant activity against ABTS and DPPH free radicals along with cytotoxicity in human gingival fbroblasts (a nontumor cell line) and oral squamous cell carcinoma cells (CAL-27).

Collection and Identifcation of Plants.
In October 2018, leaves of M. tenella were gathered in the city of Magalhães Barata, PA, located at a latitude of 00 °47′38″ S and a longitude of 47 °35′55″ W. Te collected sample, identifed as MG 237466, was ofcially registered and is now housed in a collection of aromatic plants within the herbarium of the Museu Paraense Emílio Goeldi in Pará, Brazil.

Physical and Chemical Characterization of the Sample.
Te plant material was cold-dried in a temperaturecontrolled room with air conditioning.Te sample was fnely ground using a knife mill (Model TE-631/3; Tecnal, Brazil).Te moisture content of the plant material was determined using a moisture analyzer (ID50, Marte Científca, Brazil) based on infrared spectroscopy.Te extraction yield of EO was calculated according to the procedure described by Santos et al. [17].

Essential Oil Isolation.
To isolate EO from M. tenella leaves, hydrodistillation was carried out using a modifed Clevenger-type apparatus, which utilized 151.42 g of the sample.Extraction was performed for 3 h at 100 °C.Subsequently, anhydrous Na 2 SO 4 was added to the EO and the resulting mixture was centrifuged to eliminate moisture.Te purifed EO was carefully stored in an amber-type glass ampoule in a refrigerated environment at 5 °C to maintain its chemical composition.

Chemical Evaluation of Essential Oil.
To identify the chemical composition, the protocols already described in a previous study were used.Te brand and model of the gas chromatograph (CG-MS and GC-FID), chromatographic column, and other procedures are available in the literature [18].A comparison of the linear retention index and mass spectra of the reference standards was performed, which aligned with the information reported by Adams [19].

Cell Culture.
In this study, CAL-27 strain (derived from human tongue squamous cell carcinoma) and human gingival fbroblasts obtained from the Cell Culture Laboratory at UFPA's Faculty of Dentistry were used.Tawed cells were cultured in 25 cm 2 fasks with DMEM-F12 medium supplemented with 10% fetal bovine serum.Te cultures were maintained at 37 °C in an incubator with 5% CO 2 .Daily monitoring and proliferation assessments were performed using an inverted microscope.All the cell manipulations adhered to biosafety standards and were performed in a laminar fow hood.Growth was observed daily, and the culture medium was refreshed every 2-3 days considering cell metabolism.Te protocols used in this study were in accordance with those previously reported by Guimarães et al. [20].
2.6.In Vitro Cytotoxicity Assay.For cytotoxicity analysis, gingival fbroblast lines and CAL-27 cells were seeded in 96-well culture plates at a concentration of 1 × 10 3 cells/well and incubated at 37 °C in a humid environment with 5% CO 2 for 24 and 48 h for adhesion and cell proliferation, respectively.DMSO (LGC Biotechnology, São Paulo, Brazil) was used as a solvent to ensure solubilization of the oily compound in the culture medium.Subsequently, the compound was diluted to 125, 250, 500, and 1000 µg/mL in the culture medium (Sigma, St. Louis, MO, USA).Te control group (no treatment) contained only the culture medium, and 0.01% DMSO was used as the solvent control to eliminate the hypothesis that DMSO could interfere with the results.Te cells were then exposed to oil for 24 and 48 h [21].

Cell Viability Analysis.
Cell viability was assessed using the MTT assay following a previously reported method [20].

Antioxidant Assays
2.8.1.DPPH Assay.Te scavenging capacity (SC) of the 2,2diphenyl-1-picrylhydrazyl (DPPH•) radical by M. tenella EO was evaluated using the method outlined by Blois [22].Trolox served as the positive control, and deionized water was used as the blank control.Te samples were assessed in quintuplicate using an ultraviolet-visible (UV-vis) light spectrophotometer (800XI, FEMTO; São Paulo, Brazil).Te percentage of inhibition was determined from the observed absorbance and calculated using the formula described in the literature [23].

ABTS Assay.
Chemical interactions between EO and ABTS •+ radicals were determined according to the procedures outlined in the literature [24,25].A UV-vis spectrophotometer (800XI, FEMTO; São Paulo, Brazil) was used at a wavelength of 734 nm.Deionized water served as the blank control, whereas Trolox served as the positive control.Te percentage of inhibition was determined using the observed absorbance values, which were calculated using the formula described in the literature [23].

Yield and Chemical Profle of the Essential Oil of Myrciaria tenella.
Table 1 shows the yield and main chemical compounds in the EO of M. tenella.Te EO yield was 0.74% (w/ w), which was lower than that found in two samples of M. tenella (specimen A: 2.1%; specimen B: 1.2%) by da Costa et al. [33].Tis diference in yield may be attributed to environmental conditions and the collection period [34].

Cell Viability.
According to the ISO 10993-5 standard, a compound is considered toxic when cell viability in its presence is less than 70% compared with the control group [40].At 125 and 250 µg/mL, the EO of M. tenella was not toxic to gingival fbroblasts during a 24-h period, as shown in Figure 1.After 48 h, despite the decrease in the percentage of viable gingival fbroblasts, it remained nontoxic at both concentrations.Tis compound maintains the viability of this cell group, which is crucial for oral tissue regeneration and repair processes.
Oral cancer, the second most prevalent malignant tumor within the head and neck region, is largely dominated by oral squamous cell carcinoma, accounting for over 90% of all cases, as highlighted by the National Cancer Institute [41].Notably, EO of M. tenella exhibited pronounced antiproliferative efects on CAL-27 cells.In particular, at 24 h, a concentration of 250 µg/mL reduced cell viability to below 70%, as shown in Figure 2. By the second day, all concentrations demonstrated toxicity, underscoring the potent antiproliferative nature of M. tenella in combating this malignant neoplasm.Te cytotoxic attributes can be ascribed to the key compounds present in EO.Oliveira et al. [42] emphasized the cytotoxic potential of (E)-caryophyllene, which is a major component in EO of M. tenella.
Furthermore, Jun et al. [43] reported the cytotoxicity of caryophyllene oxide, another major compound in the EO of M. tenella.Caryophyllene, a natural sesquiterpene present in plants, exhibits potent anticancer efects and enhances drug efcacy.BCP, a phytocannabinoid, binds strongly to CB2 and lacks CB1 afnity.BCPO, an oxidative derivative, is independent of the endocannabinoid system.BCPO alters cancer-related pathways and selectively activates CB2, making BCP a potential natural analgesic.Te correlation between anticancer and antioxidant properties highlights a crucial interplay in disease prevention and treatment.Antioxidants, by neutralizing harmful free radicals and reducing oxidative stress, contribute to lowering the risk of cancer development.Moreover, many natural compounds exhibiting potent antioxidant activity also demonstrate anticancer efects, suggesting a shared mechanism.Tese dual functionalities underscore the potential of antioxidantrich diets and therapies in combating cancer and promoting overall health [42,43].Its dual anticancer and analgesic roles infuence chemotherapy efcacy and are valuable in oncology [44].Giacomo et al. [45] highlighted the anticarcinogenic activity of both caryophyllene oxide and (E)caryophyllene against human colorectal adenocarcinoma (Caco-2) and T-cell leukemia (CCRF/CEM), which resulted in a signifcant reduction in cell viability.Tese fndings collectively underscore the potential therapeutic signifcance 4 Journal of Food Biochemistry of EO of M. tenella and its major constituents, particularly (E)-caryophyllene and caryophyllene oxide, in combating oral squamous cell carcinoma [46,47].

Antioxidant Potential.
Figure 3 shows the antioxidant potential of EO of M. tenella against the ABTS •+ and DPPH • radicals.Te SC of the EO was defned as the decrease in the concentration of radicals.Trolox was used as a positive control.Te benefts of EO in the medical and food felds can be attributed to their antioxidant properties [48].Terefore, the antioxidant activity of EO of M. tenella was assessed via ABTS •+ and DPPH • radical scavenging assays [49].Te EO of M. tenella exhibited a strong SC (75%) in the DPPH assay and good antioxidant activity (28%) in the ABTS assay compared with the Trolox standard.Moraes et al. [50] reported that the EO of M. foribunda exhibited excellent scavenging activity for both ABTS •+ and DPPH • radicals.Tis result was attributed to the high levels of oxygenated monoterpene 1,8-cineole in the EO.Te EO of M. tenella primarily comprises hydrocarbon sesquiterpenes with 33.95% (E)-caryophyllene content.In addition, the diferences in the antioxidant activities of the EO of M. tenella in the DPPH and ABTS assays may correspond to the synergistic interactions between their components.Tis difference was partially attributed to the reaction medium (ethanol for the DPPH assay and phosphate-bufered saline for the ABTS assay) and reaction time (5 min for the ABTS assay and 30 min for the DPPH assay).All these factors facilitate radical scavenging kinetics [51,52].Morávek et al. [53] found that small compounds, such as volatile compounds in EO of M. tenella, exhibited a greater binding afnity for the minor groove of DNA, aligning with our docking study results.Tis phenomenon could be partially attributed to the molecular size of the ligands, which renders the formation of a stable complex with the major groove of DNA difcult.However, the compounds exhibited only a few chemical interactions with the minor groove of the DNA.In this study, these interactions were sufcient to form a stable and energetically favorable complex, according to the binding afnity energy results reported by Cascaes et al. [54].Te observed chemical interactions were hydrophobic, and electrostatic interactions were not observed because of the absence of polarizable and ionizable groups in the ligands (Figure 4).Te lowest-energy complexes from the docking results were selected as the starting point for the MD simulations, in which the complexes exhibited favorable binding energy values (Table 2).Van der Waals interactions signifcantly contributed to the formation of these complexes in addition to the favorable contributions of electrostatic and nonpolar interactions.

Conclusions
In this study, the essential oil (EO) of M. tenella has revealed signifcant advancements on various fronts.Te identifcation of the EO's chemical profle highlighted its main components, especially hydrocarbon and oxygenated sesquiterpenes, such as (E)-caryophyllene, δ-cadinene, caryophyllene oxide, and viridiforene.Additionally, its antioxidant activity was demonstrated against DPPH and ABTS radicals, indicating its relevance as an antioxidant agent.evaluation of its cytotoxicity in tumor and nontumor cells revealed a concentration and test period dependency, with promising results in gingival fbroblast cells, suggesting its potential utility in treating oral cavity-related diseases.Te in silico analysis underscored the ability of the EOs' major constituents to interact with molecular targets, corroborating its cytotoxic activity.Tese fndings indicate that the EO of M. tenella holds therapeutic potential, although further investigations are necessary to validate its efcacy and safety in clinical applications.In summary, this research represents a promising step toward the development of economically viable and accessible therapeutic alternatives.

Table 1 :
Chemical compounds identifed in the species in the essential oil of Myrciaria tenella.

Table 2 :
Binding energy values (∆G bind ) of the drug-receptor systems.