Phytochemical Analysis of the Fruit Pulp Extracts from Annona crassiflora Mart. and Evaluation of Their Antioxidant and Antiproliferative Activities

Annona crassiflora Mart., the marolo fruit of the Cerrado biome, is one of the most frequently consumed species from the Brazilian Midwest. This study aimed to evaluate the chemical composition and the antioxidant and cytotoxic properties of the fruit pulp of A. crassiflora collected at Chapada das Mesas, Maranhão, Brazil. The volatile concentrate was identified as mainly ethyl octanoate, ethyl hexanoate, and methyl octanoate. From the ethanol (LFP-E) and ethyl acetate (LFP-A) extracts were identified phenolic acids (p-coumaric, gallic, quinic, and ferulic), flavones and derivatives (apigenin, epicatechin, 2′-5-dimethoxyflavone, 3′,7-dimethoxy-3-hydroxyflavone, kaempferol-3-O-glucoside and 3-O-rutinoside, quercetin-3-O-glucoside, procyanidin B2, and rutin), aporphine alkaloids (xylopine, stephagine, and romucosine), and acetogenin (annonacin). For the LFP-E and LFP-A extracts, the total phenolic compound values were 15.89 and 33.16 mg GAE/g, the flavonoid compound content values were 2.53 and 70.55 mg QE/g, the DPPH radical scavenging activity showed EC50 values of 182.54 and 57.80 µg/mL, and the ABTS radical activity showed TEAC values of 94.66 and 192.61 µM TE/g. The LFP-E extract showed significant cytotoxicity and cell selectivity for the U251-glioma strain, presenting a GI50 value of 21.34 µg/mL, which is close to doxorubicin (11.68 µg/mL), the standard chemotherapeutic drug. The marolo fruit seems to be a promising source for developing innovative and healthy products for the food industry.


Introduction
Annonaceae has been listed among the most diversified families due to its heterogeneity and abundance in the world's tropical forests. It is a pantropical plant family of ca. 2450 species of trees and lianas, well represented in the neotropical flora with ca. 950 species. The Annonaceae comprise 26 genera and ca. 260 species in Brazil, known for their edible fruits and medicinal properties, where Annona, Guatteria, and Xylopia are the most common genera [1,2]. Some paper reviews have highlighted the traditional medicinal uses, phytochemical and pharmacological studies, and toxicity of most Annonaceae species, which indicate the presence of bioactive compounds exhibiting antimicrobial, insecticide, antiparasitic, pesticide, vermicide, and cytotoxic properties, among others [3,4]. Annonaceae are significant from an economic point of view given the numerous ways of using their species, such as fruits in food and cooking, rich in lipids and carbohydrates, in addition to diterpenes, acetogenins, alkaloids, and essential oils, among the constituents

Plant Material and Extractions
Semi-ripe fruits of Annona crassiflora were collected at Parque Nacional Chapada das Mesas (7 • 20 16" S/47 • 28 04" W), Carolina, Maranhão, Brazil, March 2016, and transported to Laboratório de Engenharia de Produtos Naturais (LEPRON), Universidade Federal do Pará, Belém, Brazil. The leaves and flowers of A. crassiflora collected for taxonomic purposes, were identified and deposited in the Emílio Goeldi Herbarium, Belém Pará state, Brazil, under number MG 222438. The lyophilized fruit pulp (LFP, 50 g) was exhaustively extracted with n-hexane (500 mL), and then the LFP residue was extracted with a hydroethanolic solution (300 mL, ethanol 70%), thereby yielding the LFP-E extract. Subsequently, the LFP-E extract was fractionated with ethyl acetate (300 mL) to furnish the LFP-A extract. The LFP-E and LFP-A extracts were dried and used for the chemical and biological tests. The lyophilized fruit pulp (10 g) was also subjected to microdistillation-extraction [25] to obtain its volatile concentrate using n-pentane (99% HPLC grade, 3 mL) (Sigma-Aldrich, São Paulo, Brazil) as the solvent.

Chemical Characterization of A. crassiflora Extracts
A clean-up step was performed to remove contaminants for the HPLC-ESI-IT-MS/MS and FIA-ESI-IT-MSn analyses. The LFP-E (hydroethanolic) and LFP-A (ethyl acetate) extracts were purified by solid-phase extraction (SPE) using Phenomenex Strata C18 cartridges (500 mg, stationary phase) that were previously activated with MeOH (5 mL) and equilibrated with MeOH:H2O (5 mL,1:1, v/v). The purified extracts were eluted from cartridges using MeOH:H2O (5 mL, 1:1, v/v), filtered through a 0.22 µm PTFE filter, and dried. Then, the extracts were diluted to 10 µg/mL using the HPLC solvent. Aliquots of 20 µL were injected directly into the HPLC-ESI-IT-MS/MS and FIA-ESI-IT-MS n .
The LFP-E and LFP-A extract analyses were performed on an HPLC-ESI-IT-MS mass spectrometer LCQ Fleet Thermo Scientific using a Kinetex C18 (4.6 × 100 mm, 100 Å and 5µm) analytical column for the LC separation. The linear gradient elution from two mixtures was used in the mobile phase: 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A gradient elution starting from 10% to 100% of B for 10 min was used with a flow rate of 1.0 mL/min. The samples emerging from the HPLC system were analyzed online by ESI-MS in the negative ion mode in series with a UV detector. For the FIA-ESI-IT-MSn analysis, the direct flow infusion of the samples was performed on a Thermo Scientific LTQ XL linear ion trap analyzer equipped with an electrospray ionization (ESI) source (Thermo, San Jose, CA, USA). A stainless steel capillary tube was used at 280 • C, a spray voltage of 5.00 kV, a capillary voltage of 90 V, a tube lens of 100 V, and a flow rate of 5 µL/mL. A full scan analysis was recorded in the range from 100 to 1000 m/z.
Multiple-stage fragmentations (ESI-MSn) were performed using the collision-induced dissociation (CID) method against the helium for ion activation. The first event was a full-scan mass spectrum to acquire data for the ions in that m/z range. The second scan event was an MS/MS experiment performed using a data-dependent scan on the [M-H] molecules from the compounds of interest at a collision energy of 30% and an activation time of 30 ms. The product-ions were then submitted to further fragmentation in the same conditions until no more fragments were observed. The identification of the different compounds in the chromatographic profiles of the hydroalcoholic (LFP-E) and ethyl acetate (LFP-A) extracts was performed by comparing their retention times and spectra with literature data.
The volatile concentrate of the lyophilized fruit pulp of A. crassiflora was submitted to GC and GC-MS analysis. It was performed on a GCMS-QP2010 Ultra system (Shimadzu Corporation, Tokyo, Japan) equipped with an AOC-20i auto-injector and the GCMS-Solution software containing standards libraries [26,27]. A Rxi-5ms (30 m × 0.25 mm; 0.25 µm film thickness) silica capillary column (Restek Corporation, Bellefonte, PA, USA) was used. The conditions of analysis were as follows: Injector temperature: 250 • C; Oven temperature programming: 60-240 • C (3 • C min −1 ); Helium as the carrier gas, adjusted to a linear velocity of 36.5 cm s −1 (1.0 mL min −1 ); split mode injection (split ratio 1:20) of 1.0 µL of the n-pentane solution; electron ionization at 70 eV; and ionization source and transfer line temperatures of 200 and 250 • C, respectively. The mass spectra were obtained by automatic scanning every 0.3 s with mass fragments in the range of 35-400 m/z. The retention index was calculated for all volatile components using a homologous series of C 8 -C 40 n-alkanes (Sigma-Aldrich, Milwaukee, WI, USA) according to the linear equation of van den Dool and Kratz (1963) [28]. Individual components were identified by comparing their retention indices and mass spectra (molecular mass and fragmentation pattern) with those existing in the GCMS-Solution system libraries [26,27]. The quantitative data regarding the volatile constituents were obtained using a GC2010 Series gas chromatograph that was operated under similar conditions to those of the GC-MS system. A flame ionization detector (GC-FID) was used to quantify the relative amounts of individual components by peak-area normalization. Chromatographic analyses were performed in duplicate. The amount of total phenolics (TP) of the LFP-E and LFP-A extracts was determined according to the Folin-Ciocalteu colorimetric procedure [29,30]. A calibration curve with gallic acid at concentrations of 1, 2, 4, 6, 8, and 10 µg/mL was prepared. The acid gallic solutions' aliquots and the samples (500 µL) were mixed with Folin-Ciocalteu reagent (250 µL, 1N) and sodium carbonate (1250 µL, 75.0 g/L). After 30 min of reaction, the absorbance was read at 760 nm in a spectrophotometer UV/Visible (Shimadzu, UV 1800, Shimadzu Corporation, Tokyo, Japan) at 25 • C and in a dark environment. The LFP-E and LFP-A extracts were solubilized in methanol at initial concentrations of 20,000 µg/mL (for the extract) and 10,000 µg/mL (for the fractions) to induce an absorbance between 0.3 and 0.7. The total phenolic content was expressed as gallic acid equivalents in mg per g of extract (mg GAE/g).

Determination of Total Flavonoids Content
The amount of total flavonoids (TF) of the LFP-E and LFP-A extracts was determined according to the aluminium chloride colorimetric procedure [31]. A calibration curve with the quercetin standard at concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 µg/mL was prepared. The aliquots of the quercetin solutions (1000 µL) were mixed with aluminum chloride (1000 µL, 2%). After 30 min of reaction, the absorbance was read in a UV/Visible spectrophotometer (Shimadzu, UV 1800, Shimadzu Corporation, Tokyo, Japan) at 420 nm in a dark environment, and at 25 • C. The LFP-E and LFP-A extracts were solubilized in methanol at an initial concentration of 6000 µg/mL. The total flavonoid content was expressed as quercetin equivalents in mg per g of extract.

DPPH Radical Scavenging Assay
The LFP-E extract was evaluated by the DPPH radical-scavenging assay [30]. DPPH is a stable dark violet free radical with a maximum absorption of 517 nm and is reduced by antioxidants. A stock solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH; 0.5 mM) was prepared in ethanol. The solution was diluted to approximately 60 µM and measured an initial absorbance of 0.62 ± 0.02 at 517 nm (Shimadzu, UV 1800, Shimadzu Corporation, Tokyo, Japan) at room temperature. The absorbance was measured at the start of the reaction, every 5 min during the first 30 min, and then at 30 min intervals until constant absorbance values were observed (plateau of reaction, 2 h). The extract (50 µL ethanol) was mixed to 1950 µL of methanolic DPPH solution (0.5 mM). The standard curves were prepared with concentrations of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (Sigma-Aldrich, St. Louis, MO, USA) of 1, 2, 4, 6, 8, and 10 µg/mL. The results were expressed as a 50% inhibitory concentration (IC 50 ) and milligrams of Trolox (mgTE/g) equivalents per gram of the extract.

ABTS Radical Cation Assay
The LFP-E extract was evaluated by ABTS radical cation assay [30,32]. ABTS radical cation was obtained by mixing 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) solution (7 mM, 5000 µL) with potassium persulfate solution (140 mM, 88 µL), followed by incubation for 16 h in the dark at room temperature. After, ABTS radical cation solution was diluted in ultrapure water until reaching the absorbance of 0.70 ± 0.02 at 734 nm (ABTS work solution). Aliquots (20 µL) of extract prepared in ultrapure water were added to ABTS work solution (2020 µL) and the absorbance was measured in a spectrophotometer (Shimadzu, UV 1800, Shimadzu Corporation, Tokyo, Japan). The results were expressed in Trolox Equivalent Antioxidant Capacity (TEAC, µMTE/g). The TEAC value was calculated by measuring the area under the curve and plotting the percentage inhibition of the absorbance as a function of time. The area under the curve was calculated for one sample dilution, which had a final percentage inhibition between 20% and 80%.

Statistical Analysis
The samples were assayed in triplicate, and the results are shown as the mean ± standard deviation. An analysis of variance was conducted, and the differences between variables were tested for significance by a Tukey test and Student's t-test. Differences at p < 0.05 were considered statistically significant. The IC 50 s values were calculated by nonlinear regression using the GraphPad program (version 5.0, Intuitive Software for Science, San Diego, CA, USA).

Composition of Fruit Pulp Extracts of A. crassiflora
The hydroethanolic (LFP-E) extract from the hydrophilized fruit pulp of A. crassiflora yielded 15.6%. The fractionation of the LFP-E extract (4 g), with n-hexane and ethyl acetate furnished the LFP-H (2.9%) and LFP-A (1.5%) extracts, respectively. The chemical profiles of the LFP-E and LFP-A extracts were analyzed using the LC-ESI-IT-MS technique (m/z 100-1000 Da) and the fragmentation data recorded are shown in Table 1. The main secondary metabolites identified were phenolic acids; flavonoid glycosides derived from quercetin, kaempferol, rutin, aporphine alkaloids; and tetrahydrofuran-type acetogenin (see Table 1 and Figure 1).   Table 1).
Compounds 1 (m/z 163), 2 (m/z 169), and 4 (m/z 193) undergo decarboxylation (loss of CO 2 , 44 Da) and correspond to p-coumaric, gallic, and ferulic acids [34,35]. Compound 3 (m/z 191), characterized by quinic acid, produced ion-fragments in m/z 173 and m/z 111, which correspond to H 2 O molecule loss and the ring-opening by a retro-Diels-Alder (RDA) mechanism [36]. Compounds 5 (m/z 269), 13 (m/z 297), and 14 (m/z 281) refer to apigenin, 3 ,7-dimethoxy-3-hydroxyflavone, and 2 ,5-dimethoxyflavone, respectively, and an RDA mechanism primarily fragmented all these flavonoid compounds [37]. The fragmentation of compound 6 (m/z 609) produced two main ion-fragments, the ion m/z 447 due to the loss of the deoxyhexose unit and the ion m/z 301 derived from the loss of the hexose-deoxyhexose unit, which is characteristic of rutin, a biflavonoid derived from quercetin with a glycosidic bond in carbon 3 of the central pyran-ring [38]. Compound 8 (m/z 593) was identified as kaempferol-3-O-rutinoside, which produced compound 7 (m/z 447), the kaempferol-3-O-β-D-glucoside, by the loss of a glycoside unit. The ion-fragment m/z 285 arose from the loss of another glycoside unit [39]. The two first fragmentations occur by the loss of the glycosyl units, thereby leaving only the structure of kaempferol that undergoes cyclization by the RDA mechanism-generating the ion m/z 152. Compound 9 (m/z 463) produced the ion-fragment m/z 301, which is related to glycoside unit loss. It was identified as quercetin-3-glycoside (isoquercetin) [40,41]. The mass spectrum of compound 10 (m/z 577) showed the ion m/z 425, which is derived from the loss of 152 Da by a retro-Diels-Alder fragmentation (RDA), the ion m/z 407 of a subsequent neutral loss of a water molecule, and the ion m/z 289, which is attributed to the interflavanic link fragmentation. The comparison between the ion-fragments allowed a structural proposal for the dimer B2-type procyanidin [42,43].
Compound 11 showed a precursor ion in m/z 289, and the subsequent fragmentation pattern, which is related to the ions m/z 271, 245, and 205, was attributed to the epicatechin. The ion of greatest intensity (m/z 245) is compatible with the molecule's enol unit loss [44]. Compound 12 (m/z 294), generated ion-fragments arising from losses of CH 3 O (m/z 264), NH (m/z 249), CH 2 O (m/z 219), and CO (m/z 191). The observed fragmentations are consistent for the aporphine alkaloid xylopine-with m/z 294. The initial loss of the mass fragments 15 and 31 Da is essential to identify whether the nitrogen of the amine group is linked to hydrogen or methyl, respectively [45]. Still, through the fragmentation analysis, it was possible to identify stephalagine (compound 15, m/z 308) and romucosin (compound 16, m/z 322), also aporphine alkaloids. The presence of such alkaloids, xylopine, stephalagine, and romucosin, was previously predicted in the extracts of A. crassiflora since they are found in several Annonaceae species and considered chemotaxonomic markers for the family [46]. The fragmentation analysis of compound 17 furnished the ions m/z 397, 379, 361, 343, 327, and 309, which are related to cleavages between the linkages C 19 -C 20 and C 15 -C 16 , and a series of fragments with H 2 O loss, thereby indicating a tetrahydrofuran ring at position C 16 -C 19 . The structure was identified as acetogenin annonacin [47,48]. Acetogenins are natural compounds isolated from Annonaceae species and derived from long-chain fatty acids (C 35 -C 37 ) via a polyketide route, which shows a γ-lactone-type terminal ring and a tetrahydrofuran-type (THF) ring along the chain, in association with oxygenated functional groups, such as hydroxyls, ketones, and epoxides [47,49].

Composition of Volatile Concentrate of Fruit Pulp of A. crassiflora
Volatile constituents are responsible for the characteristic aroma and flavor of fruits and are present in a wide range of concentrations, which are represented by different chemical classes. The constituents of the volatile concentrate in the fruit pulp of A. crassiflora were analyzed by GC-FID and GC-MS and are listed in Table 2. The yield of volatile concentrate was 0.4%. In total, 25 constituents were identified comprising more than 94% of the volatile concentrate. The primary components were ethyl octanoate (34.8%), ethyl hexanoate (29.4%), and methyl octanoate (18.3%), which comprised 82.5% of the volatile concentrate. The odor description of ethyl hexanoate (fruity, sweet), ethyl octanoate (fruity, floral), and methyl octanoate (fruity, green) point them to a significant contribution to the characteristics of the "marolo" fruit aroma, and correspond to about 90% of the total identified compounds. These results agree with previous reports, which found that esters were more abundant in the lyophilized and fresh A. crassiflora fruit, thereby justifying their characteristics as essential compounds derived from the plant lipid metabolism [12,52]. A similar result regarding the abundance of ester derivatives was obtained from the analysis of the volatile concentrate of Caryocar brasiliense Cambess.(pequi) (Caryocaraceae), another vital fruit of the Brazilian Cerrado, where ethyl hexanoate and ethyl octanoate are also the main volatile constituents [53].

Total Phenolics Content
The Folin-Ciocalteau assay allowed the estimation of flavonoids, anthocyanins, and other phenolic compounds present in the LFP-E and LFP-A extracts. The total phenolic content (TPC) for the LFP-E and LFP-A extracts were 15.89 ± 0.11 mg GAE/g dw and 33.16 ± 1.1 mg GAE/g dw, respectively. According to Vasco and coworkers [54], fruits extract are classified into three categories based on their total phenolic content: low (<1 mg GAE/g), medium (1-5 mg GAE/g), and high (>5 mg GAE/g). Therefore, the LFP-E and LFP-A extracts can be considered excellent sources of phenolic compounds. The results for the phenolic compound content in the LFP-E and LFP-A extracts of A. crassiflora fruit pulp were lower and similar to fruits collected in Goiânia, Brazil, which displayed a content of 31.08 ± 1.23 mg GAE/g [55]. However, the samples displayed higher TPC levels compared to fruits collected in Brasília, Brazil (5.80 ± 1.43 mg GAE/g) and Minas Gerais, Brazil (2.11 ± 0.60 mg GAE/g) [56,57].
The variation in the levels of total phenolic compounds analyzed in a given sample compared to the results in the literature may be due to the geographic and environmental conditions of the region of origin, physiological and genetic factors of the plant, preparation and storage conditions, and extraction method [54].

Total Flavonoids Content
The estimation of the flavonoid compounds present in the LFP-E and LFP-A extracts was quantified based on the standard curve prepared with the compound quercetin. The flavonoid content for the LFP-E and LFP-A extracts were 2.53 ± 0.51 mg QE/g and 70.55 ± 0.5 mg QE/g, respectively. The total flavonoid content of the LFP-E (2.53 mg QE/g) extract was much lower than that obtained in a previous study, which was collected in Minas Gerais and displayed amounts of 40.0 ± 0.15 mg QE/g [58]. On the other hand, the content observed in the LFP-A extract was much higher than samples previously (0.631 and ±0.04611.64 mg QE/g) [10,56]. According to the literature, flavonoids are the predominant class of phenolic compounds present in the fruit pulp of A. crassiflora [59,60]. These data are also related to the higher antioxidant activity evaluated in the reducing power tests of DPPH and ABTS reagents.

Antioxidant Activity by the DPPH and TEAC/ABTS Assays
Phenolic compounds exhibit various functional properties due to their antioxidant capacity, acting as reducing agents, hydrogen donors, transition metal chelators, reactive oxygen and nitrogen species (ROS/RNS) quenchers, enzyme inhibitors involved in oxidative stress, and regulators/protectors of endogenous defense systems [51]. The DPPH and TEAC/ABTS assays have been used to evaluate the antioxidant activity of extracts of food matrices. DPPH is applied to evaluate hydrophilic and lipophilic compounds, while TEAC/ABTS is widely used to determine the antioxidant activity of hydrophilic compounds. Both methods are based on sample antioxidants' ability to reduce the radicals by electron transferences and/or hydrogen atoms measured by absorption, which decrease at 517 nm (DPPH) and 734 nm (ABTS) [61].
Regarding the ABTS assay, the LFP-A extract presented higher and lower antioxidant activities than other extracts of A. crassiflora, whose values were 131.58 ± 19.61 µM TE/g [63] and 231.79 ± 8.65 µM TE/g [51], respectively. In general, the antioxidant activity of A. crassiflora fruit pulp, determined in this work by the DPPH and TEAC/ABTS methods, is lower when compared with the results obtained for the fruit peel and seed, where there is a more significant presence of phenolic compounds: DPPH (peel: 1065.0 ± 4.45 µM TE/g; seed: 917.0 ± 7.76 µM/TE/g) and TEAC/ABTS (peel: 2022.13 ± 0.98 uM TE/g; seed: 190.54 ± 7.54 µM TE/g) [64].

Antiproliferative Assay
The evaluation of the antiproliferative activity of the LFP-E extract from the fruit pulp of A. crassiflora was carried out in six human tumor cell lines: U251 (brain), MCF-7 (breast), PC-3 (prostate), OVCAR-3 (ovary), HT29 (colon), and HEP-G2 (liver), and one non-tumor cell line, HaCaT (skin) (Table 3, Figure 3).  The results presented high values for 50% growth inhibition (GI50) in most tumor cell lines, demonstrating the low cytostatic activity. Except for the glioblastoma strain (U251), in which the LFP-E extract showed a significant antiproliferative effect, presenting a GI50 value of 21.34 µg/mL, close to that obtained for doxorubicin (11.68 µg/mL), the chemotherapeutic drug widely used to treat different types of cancer, and, in this work, was used as the positive control. Furthermore, LFP-E was at least 21 times less toxic for the nontumor cell line (HaCaT) than doxorubicin, with GI50 values of >100 µg/mL and 4.79 µg/mL, respectively. The tumor cell selectivity is an essential factor for possible drugs since they The results presented high values for 50% growth inhibition (GI 50 ) in most tumor cell lines, demonstrating the low cytostatic activity. Except for the glioblastoma strain (U251), in which the LFP-E extract showed a significant antiproliferative effect, presenting a GI 50 value of 21.34 µg/mL, close to that obtained for doxorubicin (11.68 µg/mL), the chemotherapeutic drug widely used to treat different types of cancer, and, in this work, was used as the positive control. Furthermore, LFP-E was at least 21 times less toxic for the non-tumor cell line (HaCaT) than doxorubicin, with GI 50 values of >100 µg/mL and 4.79 µg/mL, respectively. The tumor cell selectivity is an essential factor for possible drugs since they exhibit low selectivity against cancer cells and can cause toxicity to normal cells (side effects) [65]. The antiproliferative activity observed for the LFP-E extract of A. crassiflora can be explained by the presence of the acetogenins annonacin and squamocin, which are compounds capable of preventing electron transport in the mitochondrial complex I through the inhibition of the NADH coenzyme, which is responsible for the production of cellular energy [23]. The LFP-A extract of A. crassiflora showed no antiproliferative effect.
Other hydroethanolic extracts of A. crassiflora leaves in a human tumor cell lineage were previously reported, with significant antiproliferative activity and GI 50 values less than 10 µg/mL for various cell lines [66,67]. Interestingly, the peels and seeds of the A. crassiflora fruit contained similar phytochemicals [64]. Also, the extracts were found to have in vitro anticancer activities against multidrug-resistant ovary adenocarcinoma, glioma, breast, non-small cell lung cancer, prostate, ovary, colon, and leukemia.

Conclusions
The present study revealed that the extracts of the fruit pulp of A. crassiflora showed significant phytochemical constituents belonging to phenolic acids, flavonoids, aporphine alkaloids, acetogenins, and fatty acid esters classes. Many of the identified compounds are correlated to the observed antioxidant and antiproliferative activities, suggesting that the extracts could be used in the future for potential applications in functional foods, oxidative stress control, and cancer treatment. Also, the odors of ethyl hexanoate (fruity, sweet), ethyl octanoate (fruity, floral), and methyl octanoate (fruity, green) contribute significantly to the characteristics of the "marolo" fruit aroma.