Eugenia uniflora L. seed and pulp extracts: phytochemical profile, cytotoxic potential, antitumoral activity, and α-amylase and α-glucosidase inhibition capacity

Abstract In this study, phytochemical profiling, cytotoxic potential, antitumoral activity, and α-amylase and α-glucosidase inhibition capacity of extracts of seed and pulp of Eugenia uniflora L. fruits were investigated. The extracts were obtained using a cellulase complex and the phenolic compounds were quantified. The cytotoxic potential and antitumoral activity were evaluated using peripheral blood mononuclear cells and melanoma-type tumor cells, respectively. The α-amylase and α-glucosidase inhibition capacity was determined. For all extracts, the compounds identified and quantified were salicylic acid, protocatechuic acid, gallic acid and, myricitrin. For extract of pulp, ellagic and p-coumaric acids were also identified and quantified. The extracts do not show cytotoxicity in peripheral blood mononuclear cells. Extract of seed was able to decrease cell viability in melanoma-type tumor cells within 24 h of exposure. The concentration 5 µg mL−1 of extracts inhibited 7.73% of α-amylase and 15.34% of α-glucosidase. The Pitanga extracts presents substances with biological activities. Graphical Abstract


Introduction
Pitangueira (Eugenia uniflora L.) is a fruit tree belonging to the Myrtaceae family, an important family of edible fruits.Fruit ripening begins when the epicarp changes from green to yellow/orange or light red, until the final color is red, orange or dark purple/ black (Franzon et al. 2018).Pitanga, fruit of the Pitangueira, is a globular, furrowed, and shiny drupe, presents eight to ten longitudinal grooves, with a generally aromatic, sweet, and acidic flavor, rich in calcium, phosphorus, magnesium, vitamins C and precursors of pro vitamin A. It usually has one or two seeds and rarely more than three, which are discarded for consumption, and correspond to about 23% of the fruit (Oliveira et al. 2017;Franzon et al. 2018).
Pitanga has bioactive substances, characterized mainly by phenolic compounds.In the pulp, flavanols, proanthocyanidins, anthocyanins and carotenoids were identified, especially lycopene, lutein, beta-carotene, rubixanthin, violaxanthin and zeaxanthin.In the seed, phenolic compounds were identified, as ellagic acid, quercetin and kaempferol.Studies demonstrate that the pulp, leaves and/or seed have a high antioxidant capacity, anti-inflammatory activity, apoptotic activity, antihyperglycemic effect and antidyslipidemic effects (Bagetti et al. 2011;Victoria et al. 2012;Denardin et al. 2014;Oliveira et al. 2014Oliveira et al. , 2017;;Chaves et al. 2018).Although the seed, is a byproduct originating from several fruits, has currently received a lot of attention in bioactivity studies (Sunil et al. 2019).It should be noted that there is a gap in the literature regarding research to characterize phenolic compounds and bioactivities of Pitanga extracts and bioactivities (Fidelis et al. 2022).In this work, we report the phytochemical profile, cytotoxic potential, antitumoral activity, and a-amylase and a-glucosidase inhibition capacity of the aqueous extracts from the pulp and seeds of E. uniflora L. fruits.

Enzymatic extraction for phenolic compounds and phytochemical profiling of extracts
After the enzyme-assisted extraction, breaking points are observed in the structures of the seed (Figure S1) and the pulp (Figure S2), which characterizes the hydrolysis of plant tissues by cellulases, enabling the transfer of phenolic compounds to the aqueous medium.The cell wall of plants has complex polysaccharides, such as cellulose, hemicellulose, pectin, and lignin.This structure, when in contact with hydrolytic enzymes, is broken and degraded (Gligor et al. 2019).In this way, the migration of phenolic compounds to the aqueous medium occurs.Table S1 shows the results of the identification and quantification of the phenolic compounds of the extract of seed (ASE) and extract of pulp (APE) obtaining by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS).For ASE, four compounds were identified and quantified, namely: gallic acid, protocatechuic acid, salicylic acid and myricitrin.For the APE, six compounds were identified and quantified, namely: ellagic acid, protocatechuic acid, gallic acid, salicylic acid, p-coumaric acid and myricitrin.In general, phenolic acids were the largest class of phenolic compounds found in the pulp and seed of Pitanga, while the only flavonoid identified was myricitrin, in both fractions.It should be noted that even with compounds in common among the extracts, the concentration differs.For gallic acid, the concentration in ASE is higher than in APE, which may interfere with possible biological activities performed by the extracts, considering the influence of each molecule.Tenfen et al. (2021) report that phenolic acids are the predominant group of phenolic compounds in species of the genus Eugenia.Phenolic acids (ellagic acid, gallic acid, p-coumaric acid) and flavonoids such as myricetin have also been identified in leaves of Pitanga (Bezerra et al. 2018;Falcão et al. 2018).Nic acio et al. (2017), found a higher content of phenolic acids (gallic, chlorogenic, p-coumaric, ferulic and ellagic acids) in the pulp of the Cereja-do-Rio-Grande (Eugenia involucrate.It was possible to confirm that the enzyme-assisted extraction in aqueous medium provides extracts that contain the phenolic compounds already reported for the seed and pulp of Pitanga.These compounds are reported as promising for drug development.

Cytotoxic potential
Peripheral blood mononuclear cells (PBMC) and melanoma-type tumor cells (SK-MEL-28) were treated with ASE and APE in different concentrations and at exposure times of 24 h, 48 h and 72 h.For PMBC, there was no toxicity when exposed to ASE (Figure S3) and APE (Figure S4), and there was an increase in cell viability when exposed to most concentrations tested, for both extracts.For SK-MEL-28, cells treated with ASE (Figure S5), in 24 h exposure, there was a decrease in cell viability, with statistically different values (p < 0.05) from the control experiment, when concentrations of 100 mg mL À1 were applied (decrease of 17.72%) and 500 mg mL À1 (decrease of 15.16%).For 48 h and 72 h of exposure, there was an increase in cell viability, with statistically different values (p < 0.05) from the control experiment for concentrations of 100 mg mL À1 , 250 mg mL À1 and 500 mg mL À1 , for others, the values remained the same as the control.For APE (Figure S6), there was an increase in cell viability, with statistically different values (p < 0.05) from the control experiment.There are gaps in the literature about which phenolic compounds present in the pulp and seed of Pitanga are related to cell viability in tumor and non-tumor cells, and the synergistic effect between the compounds may be related to the increase or reduction of cell viability.

Melanoma tumor cells migration assay exposed of ASE and APE
The wound healing method was used to determine migration of SK-MEL-28.The method is based on the observation that, after creating a 'wound' in a confluent cell monolayer, the cells at the edge of that wound will move towards the opening, until new cell-cell contacts are established again (Liang et al. 2007).The images of wounds in the cell monolayer before (control) and after exposure to ASE (Figure S7) and APE (Figure S8) for 24 h and 72 h, demonstrate that there was inhibition of cell migration in most concentrations tested, mainly when the cells were exposed for 24 h to the extracts.Many bioactive compounds are shown to possess inhibitory mechanisms at different stages of carcinogenesis (angiogenesis, metastasis, migration, invasion, and inhibition of cell cycle phase), leading not only apoptosis, but changing the cellular signaling pathways (Strickland et al. 2015).

A-Amylase and a-glucosidase inhibition capacity of ASE and APE
Table S2 show the capacity of the extracts (ASE and APE) to inhibit of the a-amylase and the a-glucosidase.The results show that concentrations of up to 100 mg mL À1 of ASE were able to inhibit a-amylase.For the APE, only the concentration of 50 mg mL À1 showed inhibition, however the percentage of inhibition of a-amylase was lower than that obtained for ASE.For a-glucosidase, it appears that for 5 mg mL À1 , 50 mg mL À1 , 100 mg mL À1 and 250 mg mL À1 of ASE, there was inhibition of the enzyme.For the APE, it appears that there was inhibition of a-glucosidase when 5 mg mL À1 was used, followed by much lower values of percentage inhibition when 50 mg mL À1 and 250 mg mL À1 were used.Tan et al. (2017), demonstrated that myricetin was the phenolic compound with the most inhibitory capacity against a-amylase, a-glucosidase, and lipase.In our study, the highest content of myricitrin was found in ASE, an extract that showed greater inhibition of enzymes.However, the amount of this phenolic compound present in the ASE may be partially responsible for the percentage values of inhibition of a-amylase and a-glucosidase below 16%.This inhibition occurs due to the ability of phenolic compounds to combine with enzymes such as a-amylase, a-glucosidase forming stable complexes (Oliveira and Silva 2017).

Experimental
The experimental section is available in supplementary material.

Conclusion
Aqueous extracts of the seed and pulp of Pitanga obtained by enzyme-assisted extraction have phenolic compounds with bioactivities.It was possible to identify and quantify four phenolic compounds in the seed and six phenolic compounds in the pulp.The extracts of the seed and the pulp did not show toxicity in peripheral blood mononuclear cells.Regarding the antitumor activity, the seed extracts were able to modify both the cell viability of melanoma-type tumor cells, as well as cell migration.The seed extracts showed inhibition of enzymes involved in carbohydrate metabolism, a-amylase, and a-glucosidase.In view of this, the extracts demonstrated promising results, in vitro, regarding an antitumoral activity and a-amylase e a-glucosidase inhibition capacity, becoming a potential raw material for possible treatments or therapeutic adjuvant.However, further studies are needed to understand how these compounds act individually and synergistically in biological activities, such as antitumoral activity and a-amylase e a-glucosidase inhibition and what are their mechanisms of action.

Disclosure statement
No potential conflict of interest was reported by the authors.