Eugenia brasiliensis: Analysis of the Chemical Profile and Evaluation of Cytotoxic Potential

ABSTRACT

This work evaluated the antiproliferative potential of Eugenia brasiliensis leaf extracts against the HeLa cervical cancer cell line. The extracts were prepared by maceration using hexane (EBH), dichloromethane (EBD), and ethyl acetate (EBAE), and they were evaluated for their antiproliferative potential through a 3‐4,5‐dimethyl‐thiazol‐2‐yl‐2,5‐diphenyltetrazoliumbromide (MTT) assay in the cervical cancer cell culture (HeLa cell line) and a non‐cancer cell line (NIH‐3T3). EBH, EBD, and EBAE were cytotoxic in HeLa cells, with 50% inhibition concentration (IC₅₀) = 97.59, 31.03, and 57.67 µg/mL, respectively. EBD inhibited migration and altered the cell cycle. Eight compounds were tentatively assigned to E. brasiliensis leaf extracts by interpreting their fragmentation patterns and molecular formulae obtained from mass spectra. The dichloromethane extract of the leaves of E. brasiliensis against the cells of cervical cancer showed potential cytotoxicity activity.

Leaves of Eugenia brasiliensis were dried, crushed and macerated with polarity increment. The antiporliferative and anti‐migratory potential and the cell cycle were evaluated. 28 compounds were tentatively identified by mass sceptrometry.

1. Introduction

Eugenia brasiliensis Lam., belonging to the Myrtaceae family, is a species native to the coastal region of the Atlantic Forest, occurring between the states of Bahia and Santa Catarina. Commonly known as grumixama or Brazilian cherry, this plant bears fruit between November and December [1, 2]. It is widely used in folk medicine as a diuretic, astringent, and antirheumatic, as well as for treating diarrhea and arthritis. Scientific studies have identified antimicrobial, antifungal, anti‐inflammatory, and antioxidant properties in different plant parts, including fruits and leaves [3, 4].

These biological properties can be attributed to the plant's chemical composition, which is rich in secondary metabolites. These compounds are produced as an adaptive response to various environmental conditions and play diverse roles, including defense against herbivores and pathogens. Among the major classes of bioactive metabolites, terpenoids, quinones, saponins, flavonoids, and isoflavones stand out, many of which have significant pharmacological relevance [5, 6].

With the development of organic chemistry in the 19th century and the possibility of isolating and identifying the compounds present in plants, it was possible to develop medicines from these secondary metabolites. Among these compounds, those that managed to treat cancer stood out, being able to induce apoptosis, inhibit cell proliferation and migration, and modulate epigenetic changes associated with tumor progression. The search for novel antitumor drugs with high efficacy and reduced toxicity to non‐tumor cells remains a significant challenge in oncology, aiming for safer and more effective treatments [6, 7].

Among the various types of cancer, cervical carcinoma stands out as one of the leading causes of female mortality, particularly in less developed regions. This cancer is predominantly caused by persistent infection with oncogenic subtypes of human papillomavirus, with types 16 and 18 accounting for approximately 70% of diagnosed cases [8, 9]. In 2018, about 34 000 deaths were recorded in the Americas due to this malignancy, making it the second leading cause of cancer‐related death among women in 36 countries [10, 11].

Although the therapeutic potential of E. brasiliensis is recognized in several areas of medicine, there is a significant gap in the scientific literature regarding the specific action of this plant on cervical cancer. Few studies have thoroughly investigated the effect of the secondary metabolites of this genus on cervical tumor cells, especially concerning antiproliferative and anti‐migratory activity. Therefore, it is essential to fill this gap by studying the chemical composition of extracts of E. brasiliensis leaves and evaluating their cytotoxic potential in cervical cancer cells (HeLa) and non‐tumor cells. The identification of bioactive compounds with antiproliferative activity could contribute to the development of new therapeutic strategies for the treatment of this malignancy.

2. Results and Discussion

2.1. Cell Viability and Selectivity

The extracts produced from the leaves of E. brasiliensis were tested on HeLa cancer cell lines, and their 50% inhibition concentration (IC₅₀) was calculated, through which graphs were obtained (Figure 1). The E. brasiliensis fractions of hexane (EBH), dichloromethane (EBD), and ethyl acetate (EBAE) obtained their respective IC₅₀ values equal at 97.59 µg/mL; 31.03 µg/mL (p < 0.0001 and R² = 0.9917) and 57.67 µg/mL (p < 0.0001 and R² = 0.9835). Doxorubicin (DOXO) and Carboplatin (CARBO) were used as positive controls and obtained an IC₅₀ of 1.10 µg/mL (p < 0.0001 and R² = 0.9944) and 52.45 µg/mL (p < 0.0001 and R² = 0.9754). The extracts were tested at 3–250 µg/mL concentrations. CARBO (3–200 µg/mL) and DOXO (0.39–25 µg/mL) were used as positive controls, while the negative control consisted of cells treated only with a Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). IC₅₀ values were determined by nonlinear regression and statistical comparisons between groups were conducted using one‐way analysis of variance (ANOVA), followed by Tukey. Results are presented as mean ± standard deviation (n = 3).

FIGURE 1.

Cell viability assays in HeLa cells. Cell viability evaluation using the 3‐4,5‐dimethyl‐thiazol‐2‐yl‐2,5‐diphenyltetrazolium bromide (MTT) assay after treatment with EBH (E. brasiliensis hexane fraction), EBD (E. brasiliensis dichloromethane fraction), and EBAE (E. brasiliensis ethyl acetate fraction). Statistical analysis was performed using GraphPad Prism software version 8.4.3. The symbol “*” indicates a concentration at which there was a statistically significant difference (p < 0.05) compared to the negative control.

The extracts EBD and EBAE were chosen to continue their studies because they had a lower IC₅₀ in HeLa. These extracts were tested against the non‐fibroblast tumors (NIH‐3T3), and then the IC₅₀ was calculated using nonlinear regression and graphs (Figure 2). The extracts were tested at 3–250 µg/mL concentrations. CARBO (3–200 µg/mL) and DOXO (0.39–25 µg/mL) were used as positive controls. In contrast, the negative control consisted of cells treated only with a DMEM medium supplemented with 10% FBS. IC₅₀ values were determined by nonlinear regression and statistical comparisons between groups were conducted using one‐way ANOVA, followed by the Tukey test.

FIGURE 2.

Cell viability assays in NIH/3T3 cells. Cell viability evaluation using the 3‐4,5‐dimethyl‐thiazol‐2‐yl‐2,5‐diphenyltetrazolium bromide (MTT) assay after treatment with dichloromethane (EBD), ethyl acetate (EBAE), carboplatin, and doxorubicin. Statistical analysis was performed using GraphPad Prism software version 8.4.3. Results are presented as mean ± standard deviation (n = 3). The symbol “*” indicates a concentration at which there was a statistically significant difference (p < 0.05) compared to the negative control (one‐way analysis of variance [ANOVA], followed by the Tukey test).

EBD and EBAE had IC₅₀ values of 32.27 µg/mL (p < 0.0001 and R² = 0.9717) and 92.70 µg/mL (n = 3, p < 0.0001 and R² = 0.9894), respectively. DOXO and CARBO were used as controls in the experiment, and their IC₅₀s were 0.78 µg/mL (R² = 0.9553) and 69.50 µg/mL (R² = 0.9157), respectively.

The ratio between the values calculated the selectivity indices (SIs) of IC₅₀ for normal and cancer cells, serving as a component fundamental in the toxicity assessment of extracts. In the literature, there are divergences regarding values considered ideal for SI. However, some authors suggest that compounds with SI > 1 demonstrate more significant toxicity to tumor cells than normal, while compound isolates with SI > 2 show better selectivity for cancer cells. There is a consensus in the literature that the higher the SI, the more toxic the sample is to cancer cells, with fewer effects on normal cells [12, 13, 14, 15].

Because of this scenario, from the data obtained in the 3‐4,5‐dimethyl‐thiazol‐2‐yl‐2,5‐diphenyltetrazolium bromide (MTT) experiments, SI was calculated for the EBD and EBAE samples, in addition to the DOXO and CARBO, which were respectively 1.03, 1.60, 0.70, and 1.32 (Table 1).

TABLE 1.

IC₅₀ values and selectivity index of the fractions of hexane (EBH), dichloromethane (EBD), ethyl acetate (EBAE), doxorubicin, and carboplatin. IC₅₀ in µg/mL, against tumor (HeLa) and non‐tumor (NIH‐3T3) lineage (n = 3).

Sample	IC50 HeLa cells (µg/mL)	IC50 NIH‐3T3 cells (µg/mL)	Selectivity Index (SI)
EBH	97.59	—	—
EBD	31.03	32.27	1.03
EBAE	57.67	92.70	1.60
DOXO	1.10	0.78	0.70
CARBO	52.45	69.50	1.32

The results obtained from the samples about the cell line HeLa tumor cells indicate that EBD extract can inhibit proliferation with a lower IC₅₀ value than EBH and EBAE. In addition, it is observed that EBD is more toxic to cancer cells than CARBO, the reference drug in the treatment of cervical cancer. Cisplatin and CARBO are some drugs commonly used to treat cervical cancer. The biggest challenges to be overcome are the toxic effects caused by these drugs. About 83.6% of patients treated with platinum‐based antineoplastic drugs end up having to deal with the severe toxic effects of these drugs and, as a result, may develop chemotherapy‐induced peripheral neuropathy (CIPN), manifesting as sensory paresthesias, dysesthesias, and hypoesthesias that can cause a significant adverse impact on daily activities [16].

In vitro studies using Schwann cells, it was possible to observe that CARBO decreased mitochondrial membrane potential (ΔΨm) and ATP production and led to demyelination, characterized by increased expression of p75NTR and reduced expression of myelin protein zero (MPZ). Schwann cell is a type of glial cell that produces the myelin that surrounds the axons of neurons in the peripheral nervous system, electrically isolating the nerves and thus allowing the rapid propagation of action potentials. Damage to these cells can lead to problems conducting nerve impulses [16]. These data indicate the possible toxic mechanism of the drug in CIPN. Ototoxicity in the form of tinnitus and hearing loss is also an undesirable effect of using CARBO [17]. Platinum derivatives can interact with mitochondria in the cochlea and vestibule, causing irreversible damage. These complexes can impair mitochondrial integrity and function, affecting ATP levels and inducing mitophagy [18]. So, finding a compound that is effective in causing the death of tumor cells, affecting, to a lesser extent, non‐tumor cells, is the goal in the development of new drugs.

The sample EBD showed a lower dose than CARBO for the same therapeutic efficacy and drugs with high potency, which means that small doses are already enough to achieve the desired effect. Others, with lower potency, need higher doses to achieve the same efficacy, which may increase the chance of adverse effects, as more compounds are free to interact with various receptors, and these interactions lead to unwanted effects. Of course, those studying need to move on to in vivo trials, but these results suggested a possibility of fewer systemic side effects. These first results indicated that EBD may be the basis for further studies.

In contrast, the EBAE extract showed an IC₅₀ value close to the medicine's. Based on the SIs obtained, it can be concluded that the EBD and EBAE samples from crude extracts involving the interaction of several compounds are more toxic to cancer cells than non‐tumor cells. The SIs are higher than DOXO's and very close to the IS of CARBO.

Studies with leaf extracts of the genus Eugenia are scarce. However, studies with Eugenia pyriformis in HeLa demonstrated an IC₅₀ of the crude extract and ethyl acetate fraction of 44.42 and 40.39 µg/mL, respectively [19]. The compound 2′,4′‐dihydroxy‐6′‐methoxy‐3′,5′‐dimethylchalcone, isolated from Eugenia aquea leaf was able to inhibit proliferation of the breast adenocarcinoma MCF7 cell line and to promote apoptosis via activation of poly(adenosine diphosphate‐ribose) polymerase protein and A549 lung cancer cells induce apoptosis through activation of the caspase cascade signaling pathway [20]. The genus Eugenia reveals promising but still little‐explored potential in investigating new substances with anticancer properties.

These data indicate that the extracts of E. brasiliensis have lightness selectivity for tumor cells since an elevated IS suggests that the compound has specific characteristics that allow for preferential interaction with targets present or more abundant in tumor cells, such as cancer‐specific receptors or signaling pathways. This can result in more targeted and less invasive therapies, optimizing treatment and preventing damage to the body overall. We can also think that with a higher SI value, therapeutic efficacy will be improved by preserving healthy tissues.

Given the results of the feasibility and selectivity tests, the sample of EBD had potent toxicity against tumor cells and an index of acceptable selectivity. On the other hand, the sample of EBAE showed higher selectivity, although it had a lower capacity to inhibit cell viability.

2.2. Cell Migration Assay

A tumor's malignancy is characterized by its capacity to move to different sites and tissues, resulting in metastases. Cell migration is essential in tumor invasiveness, allowing cells to reach the bloodstream and lymphatic vessels [21, 22]. The migration trial cell is widely used to evaluate cell migration in vitro and is considered the standard for this type of analysis [23, 24].

In the present study, the cell migration assay was used in the lineage HeLa in the scratch wound healing model, using the EBD, DOXO (positive control), and vehicle (negative control). The doses used for the cell migration assay, 5 and 6 µg/mL, were chosen since, in the cell viability assay, these doses did not differ statistically from the negative control in the cell viability assay, thus not inducing cell death. From the analysis of the photos taken of each well at different times, the percentage of migration‐free area was calculated in each treatment used. The EBD6 sample was taken at a concentration of 6 µg/mL and, at T1 times (12 h), T2 (18 h), and T3 (24 h), obtained, respectively, 70.50%, 68.36%, and 66.94% migration‐free area. The EBD5 sample was taken with 5 µg/mL concentration and, at the respective times, 63.00%, 56.87%, and 57.14% of free area of migration (Figures 3 and 4).

FIGURE 3.

Statistical results of the cell migration assay. It used analysis by one‐way analysis of variance (ANOVA), followed by Tukey's test, being considered *p < 0.05. EB5 (E. brasiliensis dichloromethane fraction, 5 µg/mL), EB6 (E. brasiliensis dichloromethane fraction, 6 µg/mL), DOXO (doxorubicin, 0.6 µg/mL), and NC (negative control, vehicle).

FIGURE 4.

Images of the cell migration assay. The EVOS XL core microscope took the photos at a magnification of 10x. EB5(E. brasiliensis dichloromethane fraction, 5 µg/mL), EBD6 (E. brasiliensis dichloromethane fraction, 6 µg/mL), DOXO (doxorubicin, 0.6 µg/mL), and NC (negative control, vehicle).

The results demonstrated that the sample presented a similar performance to DOXO. When we compared the EB5 and EB6 samples with the chemotherapy drug DOXO in T3, we found that EB5 had a statistically higher activity difference (p = 0.0180), and EB6 had better activity than the chemotherapy drug in the doses used (p = 0.0013). Demonstrating the potential of samples to alter cell migration. EBD, by inhibiting the migration of cancer cells, can reduce the process of tumor invasiveness and, consequently, be essential to containing the progression of the disease, improving the prognosis of treatment. These data corroborate the literature, which demonstrates the potential for preventing cell migration of different cancerous lineages by species of the Myrtaceae family, such as Syzygium cumini extracts, which have shown activity against colorectal cancer and ovarian cancer lineages [25, 26].

2.3. Flow Cytometry

Cancer cells have mutations, and the cell cycle control pathways, such as apoptosis and cell cycle arrest, may be compromised, leading to uncontrolled cell divisions. However, even in the presence of these genetic alterations, inducing cell cycle arrest may constitute an effective strategy to inhibit these uncontrolled divisions [22, 27]. The flow cytometry assay analyzes cell cycle arrest caused by tumor cell treatment. The EBD and DOXO samples were applied to HeLa cells at concentrations of previously calculated IC₅₀ values. A negative control was used for the DMEM medium. The EBD showed a statistically significant difference when compared to the NC only in the G0/G1 phase, in which 58.80% of the NC cells were found, 43.71% of those treated with Doxo, and 63.26% of those treated with EBD (Figure 5).

FIGURE 5.

Flow cytometry test for cycle arrest assessment cell line. HeLa cells were treated with vehicle (negative control), Doxorubicin (positive control), and EBD. Of the cells treated with EBD, 63.26% are in the GO/G1 phase, 4.72% in G2/M, 26.51% in the S phase, and 4.29% in the subphase G0. The analysis was performed by One‐Way ANOVA, followed by Tukey's test, and considered *p < 0.05.

The sample tested did not present a statistically significant difference in the other phases. In the flow cytometry method, it is impossible to differentiate cells in the G0 phase from those in the G1 phase; therefore, it is said that the arrest occurs in the G0/G1 phase [28]. The results indicate that the EBD sample can induce cell cycle arrest in the G0/G1 phase.

These results suggest that the sample exhibits an antiproliferative action, preventing the occurrence of late G1 phase processes that lead to the beginning of cell replication, such as the formation of the pre‐RC complex and the separation of centrioles. This blockage inhibits entry into the cell cycle and the proliferation of tumor cells. Furthermore, the cells may remain quiescent until death, thus exerting an antitumor action. According to literature data, other species of the Myrtaceae family, such as Syzygium aromaticum and Eugenia dysenterica, have also demonstrated the ability to induce cell cycle arrest in the G0/G1 phase in different tumor cell lines, respectively, using the species extract and isolated flavonoid compounds [29, 30].

Currently, drugs already used to treat various types of cancer, such as Abemaciclib and Raltitrexed, also cause cell cycle arrest in the G0/G1 phase by altering the expression of proteins and enzymes that regulate the cell cycle [27, 31]. Thus, the data collected demonstrate the antitumor potential of E. brasiliensis.

2.4. Phytochemical Profile

2.4.1. Analysis of Total Phenolic and Flavonoid Content

Phenolic compounds are secondary substances produced by a wide variety of plant species. They are characterized by structures containing aromatic rings linked to hydroxyl groups, which give them antioxidant properties. These compounds can be classified into two large groups: flavonoids and non‐flavonoids [32].

EBD and EBAE were analyzed to quantify total phenolics and flavonoids. Gallic acid and Quercetin were used to construct the analytical curve. The results obtained demonstrate a high concentration of total phenolic and flavonoid compounds in the extracts, being, respectively, 144.38 ± 16.17 µg GAE/mg (EBD), 93.48 ± 9.72 µg GAE/mg (EBAE), 32.45 ± 4.27 µg QE/mg (EBD), 24.24 ± 1.73 µg QE/mg (EBAE), according to Table 2.

TABLE 2.

Quantification of total phenolics and flavonoids in the samples. The results are expressed respectively in µg of Gallic Acid equivalent per mg of crude extract and µg of Quercetin Equivalent per mg of extract.

Sample	Total soluble phenolic (µg GAE/mg)	Total flavonoid (µg QE/mg)
EBD	144.38 ± 16.17	32.45 ± 4.27
EBAE	93.48 ± 9.72	24.24 ± 1.73

2.4.2. High‐Performance Liquid Chromatography‐Diode Array Detection‐Electrospray Ionization‐Tandem Mass Spectrometry Analysis

High‐performance liquid chromatography‐diode array detection‐electrospray ionization‐tandem mass spectrometry (HPLC‐DAD‐ESI‐MS/MS) determined the phytochemical profile of EBD and EBAE in positive and negative modes. These compounds revealed ultraviolet (UV) spectra with maximum absorption (λ_max.) on 240–280 nm, characteristic of flavanone derivatives [33]. Figure 6 shows the UV chromatogram for these fractions recorded at 270 nm.

FIGURE 6.

Ultraviolet (UV) chromatogram of CH₂Cl₂ (A) and EtOAc (B) fractions from E. brasiliensis as recorded at 270 nm.

The interpretation of the molecular ions, their fragmentation pattern, and molecular formula information obtained from the mass spectrum allowed the tentative identification of ten main compounds. Table 3 describes the retention times, mass spectrum data, and compounds tentatively assigned using positive and negative ionization.

TABLE 3.

Compounds tentatively identified in Eugenia brasiliensis by high‐performance liquid chromatography‐diode array detection‐electrospray ionization‐tandem mass spectrometry (HPLC‐DAD‐ESI‐MS/MS).

Peak no.	t R (min.)	m/z [M−H]−	m/z [M–H]+	Molecular formula	Calcd.	Error (ppm)	MS/MS	Tentative assignment	Fraction	Reference
1	1.7	191.0554		C7H12O6	191.0561	3.8	173.0954;127.0367;	Quinic acid	EBD	[29]
2	10.0	300.9984		C14H6O8	300.9990	2.1	283.9945; 257.0011; 229.0134; 201.0151	Ellagic acid	EBAE	[30]
3	11.6		393.1531	C20H24O8	393.1544	3.4	153.0558; 105.0740	NI	EBAE
		391.1395			391.1398	1.0	229.0862; 137.0243
4	17.3		387.1649	C18H26O9	387.1650	0.1	303.1221; 285.1111; 249.1139; 237.1107; 219.1012;181.0511; 147.0468	NI	EBAE
		385.1505			385.1504	−0.2	295.1188; 265.1073; 235.0970; 223.0968; 193.1225; 167.0343
5	22.6	255.0651		C15H12O4	255.0663	4.6	213.0553; 211.0733; 185.0584; 171.0440; 151.0052; 145.0640	Pinocembrin	EBD/EBAE	[31, 32]
6	26.2	269.0824		C16H14O4	269.0819	0.2	241.0834; 227.0699; 225.0911; 165.0174;	Alpinetin isomer	EBD/EBAE	[33]
7	27.2	269.0811		C16H14O4	269.0819	3.2	241.0831; 227.0690; 225.0895; 165.0178;	Alpinetin isomer	EBD/EBAE	[33]
8	28.4	269.0804		C16H14O4	269.0819	5.7	241.0836; 227.0684; 225.0878; 165.0179;	Alpinetin isomer	EBD	[33]
9	29.4		285.1123	C17H16O4	285.1121	−0.7	181.0477; 131.0497; 103.0564	Stercurensin isomer	EBD	[34]
10	32.1	285.1119		C17H17O4	285.1132	4.8	179.0322; 166.0248; 153.0522; 151.0378; 138.0301	Myrigalone H	EBD/EBAE
			287.1282	C17H18O4	287.1278	−1.3	207.0810; 191.0729; 167.0709; 105.0738; 91.0580			[35]

About the annotation of compounds, peak 1 (1.7 min, C₇H₁₂O₆) was tentatively assigned as quinic acid due to the observed precursor ion at m/z 191.0561 [M–H]^– and the fragmentation pattern, which was observed the same as the standard present by Tambara et al. [34]. Peak 2 (10.0 min, C₁₄H₆O₈) was attributed as ellagic acid, once was observed precursor ion at m/z 300.9984 [M–H]^–, and the product ions at m/z 283.9945 [M–H–H₂O]^– and 257.0011 [M–H–CO₂]^–, related to the loss of H₂O (‐18 Da) and CO₂ (‐44 Da), respectively. The fragmentation pattern was the same as that observed by data [35], which identified ellagic acid by comparing it with its commercial standard. The precursor ion at m/z 255.0651 [M–H]^– was annotated as pinocembrin (peak 5, 22.6 min, C₁₅H₁₂O₄), which is an unsubstituted B‐ring flavanone and produces characteristic fragments with loss of C₂H₂O (‐ 42 Da) and CO₂ (‐44 Da), that were observed as fragment ion at m/z 213.0553 [M–H–C₂H₂O]^– and m/z 211.0733 [M–H–CO₂]^–, respectively [36, 37]. Despite the different retention times, peaks 6 (26.2 min, C₁₆H₁₄O₄), 7 (27.2 min, C₁₆H₁₄O₄), and 8 (28.4 min, C₁₆H₁₄O₄) showed the same value for the precursor ion at m/z 269.0824, 269.0811, and 269.0804, respectively, and the fragmentation pattern was quite similar. These peaks were assimilated as alpinetin isomers, once fragments associated with the loss of C₂H₂O (‐42 Da), CO₂ (‐44 Da), CO (‐28 Da), and loss of ring B by Retro Diels‐Alder fragmentation (‐104 Da) were observed as presented by Zhao et al. [38]. Peak 10 (32.1 min, C₁₇H₁₈O₄), assimilated as Myrigalone H presented a precursor ion at m/z 285.1119 [M–H]^– on negative mode, and the pattern of fragmentation was the same observed in literature data [39]. Related to the positive mode, a precursor ion was detected at m/z 287.1282 [M + H]⁺, making it possible to observe product ions associated with the cleavage between α‐ and β‐carbon to the carbonyl group (m/z 207.0810 or 167.0709), characteristic of the aglycone dihydrochalcones structures [40].

In studies on the phytochemical composition of extracts from E. brasiliensis, the abundant presence of phenolic compounds such as catechins, flavonoids, anthocyanins, ellagitannins, catechins, ellagic acid, and carotenoids was reported. Specifically, gallic acid, quercetin, and rutin were identified in extracts from the leaves of E. brasiliensis, but studies are still scarce [41]. In an analysis of the results of the experiments in this work, it is possible to suggest that the high concentration of these secondary compounds in the sample is associated with their promising performance, with high toxicity to tumor cells and antiproliferative potential, especially in the EBD sample. This occurs because studies indicate that polyphenols have anticancer properties since they influence signal transduction processes in the immune system, activate apoptosis mechanisms, and protect cells against oxidative stress [42].

When we analyze the compounds annotated in mass spectrometry, we can corroborate data from the literature with the results obtained in this work. In silico studies, it was observed that quinic acid (QA) may interact with essential proteins in cervical cancer cells as a multitargeted compound [43]. In other studies, this compound has also shown great anticancer potential. QA promotes apoptosis in oral cancer cells by downregulating the expression of anti‐apoptotic genes and attenuating the expression of cyclin D1 and Akt signaling pathway and shows a synergistic effect with cisplatin in these cells, so QA inhibits cell proliferation and promotes apoptosis in oral cancer cells (SCC‐4) alone and with cisplatin [44].

Another compound related to the biological activity of E. brasiliensis is ellagic acid (EA), a natural phenolic constituent. In vitro and in vivo experiments have revealed that EA elicits anticarcinogenic effects by inhibiting tumor cell proliferation, inducing apoptosis, breaking DNA binding to carcinogens, blocking virus infection, and disturbing inflammation, angiogenesis, and drug‐resistance processes required for tumor growth and metastasis, thus demonstrating a compound with great pharmacological potential that can contribute to the activity of the extract [45]. Pinocembrin is a less studied compound than the previous phenolic acids, but there is already data in the literature on its activity in cancer. Pinocembrin, a bioflavonoid, is one of the primary pigments in plants that perform many biological activities. One of the studies showed that Pinocembrin induced loss of mitochondrial membrane potential with subsequent release of cytochrome C and processing of caspase‐9 and ‐3 in colon cancer cell line HCT 116. Processing of caspase‐8 was minimal. The initial trigger for mitochondrial apoptosis is translocating the cytosolic Bax protein to mitochondria [46].

 Another compound noted was alpinetin, a flavonoid component of multiple edible and medicinal plants with a wide range of biological and pharmacological activities. Recent studies demonstrated alpinetin as a bioactive dietary nutraceutical with promising anticancer activity in various human cancers through multiple mechanisms. These results showed the importance of further conducting pre‐clinical and clinical trials to develop alpinetin into a lead structure for oncological therapy [47]. Stercurensin (2′,4′‐dihydroxy‐3′‐methyl‐6′‐methoxychalcone) is a chalcone and has already been isolated from another Myrtaceae. This compound was bioassay‐guided fractionation of the methanolic extracts of the pulp and seeds of the fruits of Syzygium samarangense. It demonstrated cytotoxic activity with IC₅₀ = 35 µM in the SW‐480 human colon cancer cell line [48]. The compound Myrigalone H was identified in the genus Syzygium [49]. However, studies on isolated substances have not yet been described in cancer.

Thus, the results obtained in this study show that the EBD has relevant antiproliferative activity, as demonstrated by the MTT assay. This assay is widely used to evaluate cell viability since it measures the capacity of living cells to reduce tetrazolium into formazan, reflecting cellular metabolic activity [50]. The significant reduction in cell viability observed suggests that the compounds present in the extract can directly interfere with cell proliferation processes.

Furthermore, the EBD sample demonstrated the ability to inhibit cell migration, a relevant aspect of tumor metastasis. Cell migration is an essential process in the progression of several types of cancer, and compounds that block this cell movement are considered promising in developing antimetastatic therapies [51].

Another highlight is the observation that the extract can induce cell cycle arrest in the G0/G1 phase, which indicates a possible interference in the cell cycle transition, blocking entry into the S phase, where DNA replication occurs. The arrest in the G0/G1 phase may be associated with the modulation of cell cycle regulatory proteins, such as cyclins and cyclin‐dependent kinases [52].

The search for new anti‐cancer treatments is one of the most significant challenges in modern medicine, mainly due to cancer's complexity and diversity [53]. Although chemotherapy, radiation therapy, and targeted therapies are used, these often have unwanted side effects and limitations in effectiveness. In this scenario, native plants, such as E. brasiliensis, have gained prominence due to their therapeutic potential, especially in the fight against cancer.

Brazil, with its vast biodiversity, is a central point in the exploration of plants with medicinal properties. Scientific studies have revealed that several native species have bioactive compounds that effectively fight cancer, offering alternatives to conventional therapies. E. brasiliensis, for example, has shown promising results in in vitro studies, showing that its extracts can have cytotoxic activity, that is, the ability to induce the death of tumor cells without affecting healthy cells. This makes it a target of interest in the search for new, more effective treatments with fewer side effects [54].

In addition, studies with native plants in Brazil contribute to medicine and promote the sustainable use of natural resources and the appreciation of local flora [55]. Research on these often unknown plants can lead to the discovery of unique anticancer molecules, resulting in innovative drugs after proper investigation and clinical trials.

The scientific literature has already demonstrated the potential of compounds derived from Brazilian plants to fight different types of cancer. Research indicates that the extracts of these plants can act by several mechanisms, such as the inhibition of cell proliferation, the induction of apoptosis (programmed cell death), and the modulation of the tumor microenvironment. These mechanisms are fundamental for developing more efficient and less aggressive anticancer therapies [56].

Therefore, the innovative study of native plants, carried out concerning the preservation of native areas, gains in science, and the development of the surrounding community, may generate income for this population in the future. Given this scenario of unbridled exploitation of natural resources, investing in studies with native plants, with a significant risk of biodiversity loss, carried out with the commitment to impact the environment, should be encouraged so that biodiversity and possible molecules with pharmacological potential are not lost.

3. Conclusions

Over the years, natural products have shown great potential for developing new medications, especially in cancer. Natural compounds can target various signaling pathways, thereby impacting the molecular activity of cells. The results presented in this study demonstrate the antiproliferative potential of E. brasiliensis, especially the dichloromethane fraction (EBD), which showed the ability to inhibit cell proliferation and cell migration and induce cell cycle arrest in tumor cells. So, this species has a high concentration of phenolic and flavonoid compounds, which may be associated with its performance. The results demonstrate the anticancer potential of E. brasiliensis, but further studies, such as in vivo assays, are needed to explore this potential, which has not yet been thoroughly investigated. We also need to focus on isolating bioactive compounds to produce new phytomedicine in the search for new pharmaceutical inputs. The results obtained indicate a promising potential of the species in anticancer activity. However, specific limitations of the study are identified, especially in understanding the mechanisms involved. Thus, the need for additional investigations to deepen the elucidation of the biological processes and ensure greater scientific robustness to the demonstrations is reinforced.

4. Experimental

4.1. Obtaining the Crude Extracts

The leaves of E. brasiliensis were collected in the city of Nova Friburgo (Rio de Janeiro) in a particular area, cultivated, and previously identified by the garden's designer. Professor Aislan C. R. F. Pascoal confirmed the species in March 2023. The leaves were dried at 45°Celsius and then crushed. Bioactive compounds were extracted through exhaustive maceration, employing a 1:3 ratio of plant material to solvent. The process was conducted with a gradual increase in polarity, sequentially using hexane, dichloromethane, and ethyl acetate, resulting in the extracts EBH, EBD, and EBAE, respectively.

4.2. Cell Viability Assay

In a 96‐well plate, HeLa (cervical cancer cells—NIH/3T3 ‐ CRL‐1658 ‐ ATCC) and NIH/3T3 (murine fibroblasts—HeLa CRM‐CCL‐2 ‐ ATCC) cells were seeded at a concentration of 3 × 10 ⁴ cells/well, passage between 18 and 24, along with DMEM cell culture medium (Gibco, Thermo Fisher, Paisley, UK) supplemented with 10% FBS (Cultilab, São Paulo, BR) and 1% penicillin/streptomycin (Pen/Strep), to be incubated in a circulating oven with 5% CO2 at 37°C for 24 h. After incubation, the cells were treated with EBD and EBAE samples at 3.5 µg/mL to 250 µg/mL concentrations and then returned to the incubator for 48 h. During the treatment, one row was treated only with the vehicle (cell culture medium) to reference the negative control of the experiment; therefore, it had 100% cell viability, and for the positive control, DOXO and CARBO.

The treatment was then removed from the microplate, and 100 µL/well of a 5 mg/mL solution of MTT reagent (Invitrogen, Thermo Fisher, Oregon, USA) was added and incubated in an oven for four h. Next, the formazan crystals that were metabolized in viable cells were solubilized by the addition of 100 µL/well of DMSO (Dimethyl sulfoxide) (Labsynth, São Paulo, BR), so that it was possible to quantify the absorbance per well at 570 nm using an Epoch‐Biotek ELISA microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).

Statistical analysis was performed using GraphPad Prism 8.4.6 software (GraphPad Software, Inc.) with one‐way ANOVA, followed by Tukey's test, with p < 0.05 as statistically significant. Cells treated with vehicles alone were considered 100% viable. Experiments related to this assay were performed in triplicate [49].

4.3. Calculation of the SI

The SI of the tested samples and the positive control was calculated by dividing the IC₅₀ of NIH/3T3 cells by the IC₅₀ of HeLa cells. Samples with anti‐cancer potential are considered selective against cancer cells.

4.4. Cell Migration Assay

In a 24‐well plate, HeLa cells were seeded, between 70 and 80 thousand cells/well, next to DMEM cell culture medium, supplemented with 10% FBS and 1% Pen/Strep, and incubated until they reached 80% confluence in an oven at 37°C and 5% CO₂. The culture medium was removed and replaced with a fetal bovine serum‐starved culture medium (0.2% FBS). After the plate was incubated for another 24 h, a vertical scratch was made on the medial surface of each well with a 1000 µL tip to remove the cell monolayer (the average risk area was 493889.5 µm²). The culture medium was removed, and the wells were washed with sterile PBS.

Then, the cells were treated with DOXO (representing the positive control) and the EBD and EBAE samples, with the previously prepared treatments with a concentration of 5 and 6 µg/mL. For the cell migration experiment, finding a dose capable of inhibiting migration without inducing cell death is essential. The doses were chosen based on the cell viability test, which did not differ statistically on cell viability; even so, previous tests were performed to find doses that did not induce cell death.

The wells were photographed using an Evos XL Core Imaging System microscope (Thermo Fisher Scientific, Oregon, USA) with a 10x objective lens before treatment application (T0) and after 12 h (T1), 18 h (T2), and 24 h (T3). To analyze the images, ImageJ software (Wayne Rasband National Institutes of Health, USA) was used to obtain the percentage (%) of the area of each scratch, with a plugin “Wound Healing Size Tool” and GraphPad Prism 8.4.3 (GraphPad Software, Inc.) was used to perform the analysis statistics through ANOVA followed by multiple comparisons, (n = 3). Experiments related to this assay were performed in triplicate [57].

4.5. Flow Cytometry

In a 6‐well plate, HeLa cells were seeded at 2 × 10⁵ cells/well for 24 h. Subsequently, the cells were treated with the EBD and EBAE samples, and DOXO was prepared at a concentration corresponding to the IC₅₀ value of each. For the negative control, the cells were treated only with the vehicle. After 48 h, cells were collected from the wells using trypsin and transferred to microtubes. They were centrifuged, resuspended, and fixed with 4.5 mL of 70% ethyl alcohol. The cells in the ethanolic solution were centrifuged again and resuspended in 1 mL of a dye solution containing propidium iodide (PI), incubated at 37°C, and protected from light exposure for 30 min so that the BD Accuri C6 Plus flow cytometer could analyze them. The histograms of the samples were analyzed using the Cell Quest Pro software. The data obtained were statistically analyzed using the GraphPad Prism program using the two‐way ANOVA test, with p < 0.05 considered significant.

4.6. Analysis of the Total Soluble Flavonoid and Total Soluble Phenolic Content

EBD and EBAE samples were weighed and solubilized in 1 mL of ethanol to obtain a final 2 mg/mL concentration. The flavonoid quercetin, from a 2 mg/mL stock solution, was serially diluted to concentrations of 200–6 µg/mL, which later served as the basis for forming the analytical curve. In a 96‐well plate, 100 µL of Milli‐Q water, 60 µL of ethanol, 10 µL of aluminum chloride (from a previously prepared 40 mg/mL aqueous solution), and 10 µL of sodium acetate were pipetted. (54.46 mg/mL aqueous solution), 20 µL quercetin (from the most diluted to the most technical solution, as prepared previously), and 20 µL of each sample to be tested. The microplate was incubated for 30 min at room temperature and finally analyzed using a spectrophotometer at an absorbance of 415 nm. At the end of the experiment, the result is given as micrograms of quercetin equivalent per milligram of extract (µg of quercetin/mg). Experiments related to this assay were performed in triplicate [47]. To evaluate the total phenolic content, based on its serial dilution at concentrations of 6–200 µg/mL, an analytical curve was prepared using the standard sample, gallic acid. The samples were then pipetted into a microplate together with Folin reagents (26 µL), Na₂CO₃ (26 µL), and Milli‐Q water (182 µL) and incubated for 2 h in an environment protected from light. Finally, the absorbance of the plate was measured at 726 nm using a spectrophotometer. The results are expressed as micrograms of GAE/mg of extract or fraction on a dry basis (µg of GAE/mg). Experiments related to this assay were performed in triplicate [19].

4.7. Analysis of the Chemical Profile of Samples by LC‐MS/MS

EBD and EBAE samples (2 mg) were dissolved in 1 mL of CH₃OH:HCOOH (0.1%), centrifuged for 10 min and injected into the LC. An ultra‐high‐performance liquid chromatography system (Shimadzu, Kyoto, Japan) coupled to a microOTOF‐Q II (Bruker Daltonics, Billerica, MA, USA) with an electrospray ion source (ESI) was used for ESI‐HRMS/MS analysis. LC separation was performed on a Shimadzu XR‐ODS C18 75 mm × 2.1 mm, 2.1 µm analytical column (Shimadzu, Kyoto, Japan). Injections of 20 µL were made using an autosampler. The mobile phase consisted of 0.1% formic acid in water (solvent A) and methanol (solvent B). The elution profile was: 0.0–16.0 min (40%–70% B); 16.0–46.0 min (70%–100% B); 46.0–50.0 min (100% B); 50.0–55.0 min (100%–40% B); 55.0–60.0 min (40% B); and a flow rate of 0.3 mL/min. The analysis parameters were: capillarity of 4.5 kV (positive mode) and 3.5 kV (negative mode), ESI in positive and negative mode, final plate offset of 500 V, nebulizer at 40 psi, dry gas (N₂) with a flow rate of 8 mL/min, and a temperature of 200°C. Collision‐induced dissociation fragmentation was achieved in auto MS/MS mode. The spectra were recorded between m/z 100–1200

Author Contributions

Giovana G. F. V. de Oliveira: methodology, investigation. Milena F. Longue: methodology, investigation. Letícia M. R. Pescinelli: methodology, investigation, writing – original draft preparation. Thiago S. Charret: software, investigation. Thalya S. R. Nogueira: software, investigation. Mariana T. M. Pereira: software, investigation. Ivo J. C. Vieira: software, funding acquisition. Lucas S. Abreu: validation, formal analysis, resources, funding acquisition. Vinicius D. B. Pascoal: formal analysis, data curation, visualization, funding acquisition. Aislan C. R. F. Pascoal: Conceptualization, formal analysis, writing – review and editing, supervision, project administration. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

de Oliveira G. G. F. V., Longue M. F., Pescinelli L. M. R., et al. “ Eugenia brasiliensis: Analysis of the Chemical Profile and Evaluation of Cytotoxic Potential.” Chemistry & Biodiversity 22, no. 9 (2025): 22, e202500429. 10.1002/cbdv.202500429

Data Availability Statement

The authors have nothing to report.