2.1 Characterization of the leaves

Although some studies have already demonstrated the effects of E. uniflora, little data in the literature presents the identification of the phytochemicals present in the plant’s leaves. In addition, the knowledge about a possible correlation between phytochemicals and the biological activities of this plant is still insufficiently evidenced because of the lack of studies (Falcao et al., 2018).

A phytochemical analysis by Brasileiro et al. (2006) demonstrated that E. uniflora leaves contain alkaloids, triterpenes, tannins, flavonoids, and anthraquinones. Furthermore, high concentrations of volatile organic compounds germacrene B (9.60%), γ-elemene (7.97%), β-elemene (9.26%), germacrene D (6.46%), γ-muurolene (5.2%) and β-caryophillene (5.17%) were found in the leaves from purple pitangueira (Mesquita et al., 2017). Furthermore, Chang et al. (2011) demonstrated curzerene presence at 85.1%, the same amount reported by Costa et al. (2009).

Notably, substance from the pitangueira leaves of Rio de Janeiro (50.2%, Melo et al., 2007), Goiás (42.6%, brilliant red fruit pitangueira) (Costa et al., 2010), Goiás (34.8%), (Peixoto et al., 2019) and Nigeria (19.7%) (Ogunwande et al., 2005) were also found to be the primary component in essential oils.

Other classes of compounds are also present but to a lesser extent (less than 20% in the leaves), such as monoterpene hydrocarbons, oxygenated monoterpenes and oxygenated sesquiterpenes (Apel et al., 2004, Costa et al., 2010). The presence of flavonoids and phenolic compounds has been reported in the E. uniflora leaves ethanolic extract as described by Fiuza et al. (2008).

Besides, Figuerôa et al. (2013) demonstrated in an experimental study using different extractors from E. uniflora leaves that the total phenolic content was similar between the different types of extracts. However, the flavonoid content showed substantial differences that can significantly influence the pharmacological properties of these extracts. Thus, it is noteworthy that the extraction method also markedly influences the phytochemical constitution of the extract (Table 1).

A recent study by Sobeh et al. (2020) showed a more comprehensive phytochemical profile of the ethyl acetate fraction (EAF) of red E. uniflora leaves, which presented an extensive variety of polyphenols, such as quercetin, myricetin, apigenin and kaempferol glucosides. This extract seems to present promising properties due to this composition. Other studies using methanol extract of the leaves of E. uniflora and LC-MS/MS chromatography have identified about 17 compounds among which are secondary metabolites: gallic acid 3-O-[6́-O-acetyl-β- D-glucoside], myricetin-3-O-β-D-glucoside, myricetin-3-O-β-D-galactoside, myricetin-3-O-[6́́-O-β-N-acetylgalacto-side], myricetin-3-O-rhamnoside, myricetin-3-O-[3́́-O-acetylrhamnoside], myricetin-3-O-[2́́-O-acetyl-α-L-rhamnoside], quercetin-3,5-dimethyl ether, ellagic acid, 2,3-hexahydroxydiphenoyl-D-glucose, gallic acid, methylgallate, 3-O-D-galloylglucose, 2-O-D-galloylglucose, gentistic acid 5-O-β-D-glucoside, gentistic acid 5-O-[6́́ -O-β-D-galloyl glucoside], valoneic acid dilactone (Sobeh et al., 2019).

2.2 Purple E. uniflora fruits

Purple pitanga is the rarest variety but has a higher amount of total phenolics and therefore higher antioxidant properties than other pitanga types (Weyerstahl et al., 1988). The purple E. uniflora fruit has approximately twice the phenolic content compared to the red type (463 ± 16 to purple and 210 ± 3 % mg galic acid (GA). 100 g⁻¹ to red fruit) This has promoted an increasing scholarly interest in this variety (Bagetti et al., 2011). The purple pitanga variety presents higher pH and higher total soluble solids and carbohydrates (Table 2). Furthermore, regarding fatty acid content, it has been described that oleic acid is present in a higher proportion than others (monounsaturated and polyunsaturated fatty acids). It has been found to have a high phenolic content in the methanolic extract, and a considerable amount of anthocyanins in the ethanolic extract has been detected (Bagetti et al., 2011). Accordingly, this can be associated with greater antioxidant capacity as demonstrated by the DPPH method (Celli et al., 2011).

The anthocyanins present in fruits such as pitanga are very promising for beneficial effects on human health. Some studies report that the intake of these compounds in humans is estimated to be 180–215 mg.day⁻¹ in the USA (Kuhnau 1976). A profile characterization of anthocyanins in the ethanolic extract of purple pitanga revealed the presence of five anthocyanins: delphinidin 3-O-glucoside (99.65 ± 1.77 mg.100 g⁻¹ lyophilized fruit), cyanidin 3-O-glucoside (512.01 ± 11.18 mg.100 g⁻¹ lyophilized fruit), pelargonidin 3-O-glucoside (2.16 ± 0.13 mg.100 g⁻¹ lyophilized fruit), cyanindin 3-O-pentoside (0.83 ± 0.07) and cyanidin derivative (5.16 ± 1.23 mg.100 g⁻¹ lyophilized fruit) (Tambara et al., 2018). These anthocyanins seem to be very stable after freezing unlike other plant constituents (Lima et al., 2005). Furthermore, the peel has been found to contain more anthocyanins, flavonoids, and carotenoids compared to the pulp: Total Anthocyanins (mg.100 g⁻¹) – 26 ± 0, Total Flavonoids (mg.100 g⁻¹) – 18 ± 0, Total Carotenoids - (µ.g⁻¹) 111 ± 2 (de Lima et al., 2002).

2.3 Red E. uniflora fruit

The general properties of E. uniflora red fruit variety are characterized by lower pH, acidity, carbohydrates and phenolic content when compared to the purple type (Table 2). According to data from Denardin et al. (2015), a red pitanga has about 5.86 ± 0.03 ug of β-carotene.g⁻¹. Carotenoids are natural pigments produced during the photosynthetic process that provides coloring for flowers and fruits. These have some ingredients that can potentially prevent cardiovascular disease and cancer. Therefore, the investigation of these compounds has expanded in recent years (Yuan et al., 2015).

One study found the concentration at 0.086 ± 0.00 mg GAE.100 g⁻¹ of ascorbic acid and the total phenolic content of the ethanolic extract at 433.84 ± 60.5 mg GAE.100 g⁻¹ fw (fresh weight) as determined by the Folin-Ciocalteu method adapted from Swain and Hillis (Denardin et al., 2015). This difference in values in relation to Table 2 can be attributed to a series of processes related to the stage of maturation at harvest, genetic variants, post-harvest management, storage, and processing conditions (Szeto et al., 2002).

It is known that there is a difference in the quantification of these compounds according to the pitanga variety. However a difference is also found when using other methods for the detection of phenolic compounds. It is noteworthy that phenolic compounds are secondary metabolites widely found in vegetables, fruits, leaves and fruits and may vary depending on soil composition, temperature, season, etc. Therefore, phenolics composition varies across different studies (Sobeh et al., 2019).

A chromatographic analysis of the ethanolic extract of the red fruit performed on HPLC-DAD (wavelength at 280 nm and 360 nm) demonstrated the presence of gallic acid and derivatives, quercetin and derivatives, quercetin-3-b-D-glucoside, quercetin-3-rhammoside, kaempeferol derivative, cyanidin-3-glucoside, cyanidin derivative and malvidin derivative (Denardin et al., 2015).

2.4 Yellow E. uniflora fruit

Few data in the literature provide information about the components present in the yellow E. uniflora variety (fruits and leaves). Souto et al. (2017) assessed the phytochemical composition of the methanolic extract of the yellow E. uniflora fruit, which included the presence of quercetin, myricetin and its derivatives: 1) In Green stage (mg.100 g⁻¹ d.w): quercetin (43.3 ± 3.4), quercetin-3-glucoside (26.4 ± 5.3), myricetin (62.4 ± 8.2), kaempferol (1.5 ± 0.4); 2) In mature stage (mg.100 g⁻¹ d.w): quercetin (20.1 ± 2.1), quercetin-3-glucoside (6.7 ± 1.8), myricetin (25.0 ± 3.6), kaempferol (ND). Additionally, the content of flavonoids with respect to the stages of development do not show significant changes with myricetin being the majority compound corresponding to 48% of the total flavonoids followed by quercetin with up to 39% of the total compared to the other flavonols.

On the other hand, the phenolic content changed depending on the development stage of the fruit. In the green maturation stage, the values obtained were approximately 35 mg GAE.g⁻¹ d.w (dry weight), in the intermediate stage approximately 25 mg GAE.g⁻¹ d.w and in the mature fruit approximately 15 mg GAE.g⁻¹ d.w. The presence of ellagic acid was also detected at the mature stage (around 485.1 mg.100 g⁻¹ d.w). In the earlier maturation stages, carotenoids such as ß-carotene and lycopene are not present. They are produced at the ripest stage of the fruit before changing to the next color (7.4 ± 3.1 µg.g⁻¹ dw; 20.5 ± 5.2 µg.g⁻¹ dw, respectively), other compounds as citric, succinic and malic acids were also found in the yellow cherry as demonstrated by Souto et al. (2017).

The lack of studies on the yellow pitanga hinders the discussion of this topic, it is evident that the preferential research on the red and purple pitanga is due to the strong presence of phenolic compounds, anthocyanins and carotenoids, since they present some photoprotective ingredients that can potentially prevent diseases related to oxidative damage. The low amount of carotenoids in the yellow pitanga can be a limiting factor for studies with this variety.

2.5 Non-volatile and volatile compounds

The constituents of plants directly influence the characteristics of their fruits. The presence of volatile organic compounds (VOCs) is responsible for the aroma and consequently the flavor of fruits. Some studies also demonstrate the influence of non-volatile compounds in the flavor of these fruits (Bai et al., 2016). Many factors can influence the presence of these compounds, such as climatic and growing conditions, in addition to the ripening stage of the fruit (Kawahata et al., 1996). Some authors used different methodologies to identify volatile and non-volatile compounds present in the constituents of pitangueira, as shown in Table 3. In addition, Fig. 2 shows the structures of the main bioactive compounds present in the constituents of pitangueira.

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Fig. 2

The chemical structures of the main bioactive compounds present in the constituents of pitangueira.

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The main limitation of these studies is that many do not report the variety of the fruit or plant from which the compound was isolated, as well as its constituents. Only one study that demonstrated the presence of non-volatile compound cyanidin-3-glucoside used liquid chromatography-mass spectrometry (LC/MS) (Brasileiro et al., 2006). Many authors used the gas chromatography–mass spectrometry (GC/MS) to isolate the volatile compounds (Oliveira et al., 2008, Soares 2014). Mesquita et al., (2017) determined the volatile profiling through solid-phase microextraction (HS-SPME), combined with gas chromatography-mass spectrometry (GC–MS).

In the different maturation stages, terpenoids (monoterpenes and sesquiterpenes) are found. However, the presence of esters only was determined in the last stages of maturation (da Silva et al., 2019). In addition, it was observed that the different varieties of E. uniflora are responsible for the variability in the volatile compounds present in the leaves of the plant. The orange¹ variety of pitanga presents a high amount of hydrocarbon monoterpenes in relation to the purple and red that present similar profile (Mesquita et al., 2017). Some compounds, such as selin-1,3,7(11)-trien-8-one, are also found in the fruit extract (Oliveira et al., 2008). The presence of selina-1,3,7(11)-trien-8-one, curzerene and atractylone, spathulenol and germacrone were identified in specifically in red-orange¹, red and purple, respectively. It has been identified that the increase in the presence of anthocyanins and tannins decrease during the maturation process of pitanga (Ramalho et al., 2019).

The differences of climate conditions can exert influence on the chemical constituents of E. uniflora. The prevalence of monoterpenes is related in the pitanga fruits, but differences in relation to majority compound are observed in fruits cultivated in Brazil and Cuba, with the presence of trans-b-ocimene (36.2%) and curzenene (38,9%), respectively (Pino and Correa 2003, Oliveira and Padilha 2007). In addition, different seasons in the same country have been shown to affect the constitution of essential oils in the leaves of the plant. In the dry season, the essential oil of the leaves of E. uniflora of the red–orange variety presented as major compounds spathulenol (10%) and caryophyllene oxide (4.1%). However, in the wet season, the main major constituent was seline-1,3,7(11)-trien-8-one epoxide (29%) (Costa et al., 2009).

4.1 Antibacterial activity

Infections caused by bacteria are a significant public health concern. Multi-resistant microorganisms are present in hospitals and in other places. The investigation of new molecules in natural products that can kill resistant microorganism species presents a plethora of possibilities. This property has been found for E. uniflora and has been explored in different scenarios.

A study evaluating the in vitro antibacterial activity of the hydroalcoholic extract of ripe E. uniflora demonstrated that it reduced the biofilm formation of Streptococcus mutants, Streptococcus oralis and Lactobacillus casei, with a reduction in bleeding caused by gingivitis, which was attributed to its anti-inflammatory action due to the presence of flavonoids (Jovito Vde et al., 2016). Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, Streptococcus mitis, and Streptococcus oralis are involved in the biofilm formation and consequently cause predisposition to cavities. The ethanolic extract of the mature and unripe fruits as well as leaves extracts showed antibacterial activity, with the exception of the essential oil derived from the leaves (Oliveira et al., 2008).

In addition, another study with the essential oil from E. uniflora leaves demonstrated antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes. These bacteria are part of the gram-positive group, whereas no effect was observed in the gram-negative pathogens (Victoria et al., 2012). Similarly, the essential oils were found to have bactericidal effect against other gram-positive pathogens, such as Bacillus subtilis, Streptococcus faecalis and Staphylococcus albus. Whereas no effect on the gram-negative group was found (Thambi et al., 2013). In gram-negative bacterias (E. coli) this oil present inhibitory effect and increase the activity of antibiotic aminoglycosides (Pereira et al., 2017).

On the other hand, positive results of E. uniflora against gram-negative bacteria were reported with ethanolic extract of the leaves, and inhibition was effective against Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa and Serratia marcescens (Table 4) (Fiuza et al., 2009). Furthermore, pitanga seed have been found to have biological properties against both gram-negative and gram-positive pathogens. The essential oil and seed extracts present similar antibacterial properties due to the presence of oxygenated groups in the structure of the isolated compounds such as terpenoids, which may be responsible for this potential (Santos et al., 2015).

E. uniflora seeds contain lecithin that binds to microorganisms such as bacteria and can inhibit the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella spp at 1.5 μg.mL⁻¹ and Bacillus subtilis, Streptococcus spp. and Escherichia coli at 16.5 μg.mL⁻¹. This effect can be explained by a channel formation in the membrane cells, which affects cell permeability and leads to cell death (Table 4) (Oliveira et al., 2008) (see Table 5).

4.2 Antifungal activity

Candida spp. is the primary fungal pathogenic agent in humans. Despite belonging to the human microbiota, alteration in tissues in association with virulence factors allow this fungus to invade patients with lower immunologic resistance that are mostly affected. It was demonstrated that E. uniflora leaf extract could reduce biofilm formation in cells isolated from the oral cavity in transplanted patients. Various Candida fungal strains were evaluated such as C. tropicalis, C. parapsilosis, C. glabrata, C. orthopsilosis, C. metapsilosis, and C. dubliniensis. A study showed that pitanga could reduce adherence of human oral epithelial cells to the biofilm formation and alter the hydrophobicity of the cell surface of Candida albicans, suggesting that the antifungal effect of E. uniflora is due to gallic acid and myricitrin, phytochemicals that can act against Candida spp per se (Souza et al., 2018).

Another study on E. uniflora essential oil also demonstrated antifungal activity against Candida spp. When this oil was administered associated with the antifungal fluconazole, the authors did not observe a synergic effect. In fact, this combination compromised the effect of the drugs, whereas the isolated use of the plant or the antifungal were effective. Despite the incompatibility with fluconazole, antifungal effect of E. uniflora essential oil were attributed to interference with virulence factors of candidiasis and morphological changes caused through the formation of filamentary structures (Dos Santos et al., 2018). However, when the ethanolic extract was associated with the antifungal drug metronidazole, a combined effect against C. tropicalis was found; however, this synergic activity was not evidenced in other strains (Santos et al., 2013). Another virulence factor of Candida spp. is the secretion of hydrolytic enzymes which can be reduced by leaf extract from E. uniflora (Silva-Rocha et al., 2015).

Besides fungus that colonize the oral cavity, there are those that affect the skin and are responsible for dermatitis. Among these organisms is Paracoccidioides brasiliensis, the main organism responsible for systemic mycosis in Latin America. Essential oil from E. uniflora was able to inhibit the fungus growth at a concentration of 62.5 µg.mL⁻¹. This effect can be attributed to the presence of selinatrienone derivatives and curzerene compounds in this oil (Costa et al., 2010).

The studies describe the inhibitory effect of E. uniflora against biofilm formation and fungal growth. The presence of some compounds in the pitanga extract, such as oleic acid, can be responsible for this response. Muthamil and collaborators (2020) found that this compound is able to reduce the filament formation, induce alterations in ergosterol present in the cell membrane of Candida spp. In addition, the acid oleic affects the expression of genes (asl-1, sap2, hwp1 and cst20) involved in the virulence response of Candida spp. The flavonols myricetin and quercetin, also present in this fruit, present effect in the biofilm formation of P. aeruginosa that acts on the phenazines, a molecules involved in the intracellular redox state and colony biofilm formation, promoting alterations in the electron transporter of this molecules (Pruteanu et al., 2020).

4.3 Antiparasitic activity

Parasitic diseases are significant and neglected health problems in emerging countries and can affect both humans and animals. Social and cultural factors are related to the parasite infections in humans caused by inadequate and unsanitary conditions. Antiparasitic drugs can be quite toxic and thus the search for natural products has been increasing (Wink 2012). Therefore, antiparasitic properties of native plants are particularly interesting for the population of less developed countries.

The essential oil from pitangueira leaves demonstrated antiparasitic activity (IC₅₀ 6.10 ± 1.80 µg·mL⁻¹) against Leishmania amazonensis (L. amazonensis), which indicates a higher efficacy than the drug pentamidine isethionate (IC₅₀ 23.22 ± 9.04 µg·mL⁻¹). This oil is rich in sesquiterpenes, which causes alterations in the mitochondrial and plasmatic membrane of the parasite (Kauffmann et al., 2017). The Leishmania genus affects various regions worldwide. In the Americas, the phlebotomus of the genus Lutzomyia is the vector. The manifestations of Leishmaniasis include cutaneous sores (localized and disseminated), muco-cutaneous, and visceral ulcers or kala-azar (Torres-Guerrero et al., 2017).

Another study with L. amazonensis showed that E. uniflora essential oil caused 100% inhibition of promastigotes at the concentrations of 400, 200, and 100 μg·mL⁻¹ after 72 h of exposure with an IC₅₀ of 1.75 μg·mL⁻¹. As the parasite infects macrophages, a study evaluated the oil’s effect in the cells infected with L. amazonensis. Pitanga essential essential oil was able to reduce parasite infection due to greater activation of macrophages, with increased phagocytosis capacity and lysosomal activity. It is believed that the terpene compounds act in the isoprenoid pathway, inhibiting one of the stages of ubiquinone biosynthesis, which is required for the advance of the parasite stages. This might explain the anti-Leishmania activity of this plant. The lipophilic constituents of essential oils derived these from plants can alter the constitution of cell barriers and lipids of the plasma membrane of the parasitic promastigote stage, leading to the release of cell contents and consequently the inhibition of cytoplasmic processes (Rodrigues et al., 2013).

Similar to Leishmania, Trypanosoma cruzi is transmitted by an insect, by blood transfusion, organ transplant or ingestion of contaminated food. The recovery rate following medicine treatment is high only in the acute phase of this disease and is less than 20% effective in the chronic phase. However, the drugs available for the treatment are highly toxic. Therefore, it is necessary to search for new alternatives with antiparasitic activity and safety. A study with the epimastigote form, found in the vector of this disease, showed that the pitanga leaf extract could inhibit 80% of the parasite at the concentration of 100 µg·mL⁻¹. This extract also showed low cytotoxic activity in J774 macrophage cells. Only 8% of the cells exposed to a concentration of 100 µg·mL⁻¹ of the extract were unviable, and this toxicity was reduced to 0% with a concentration of 10 100 µg·mL⁻¹. These results indicate that E. uniflora has anti-epimastigote potential and locytotoxicity (Costa et al., 2010).

The efficacy of the plant was evaluated also against other parasites of the genus Trypanosoma (T. brucei), responsible for anemia and damages to organs, such as liver and kidney. The leaves of E. uniflora demonstrated effectiveness against trypanosomal infection and the symptoms of this condition, such as anemia. The enzyme adenosine kinase (TbAK) was a target of the plant action (Abdelfattah et al., 2021). In addition, de Souza et al., (2017), demonstrated that components such as tannins and flavonoids of E. uniflora fractions are able to protect against the promastigote and amastigote forms of Leishmania spp.

Another study evaluated the effectiveness of the extract obtained from the E. uniflora leaves against the parasite Trichomonas gallinae, which is responsible for trichomoniasis that affects birds. However, the methanolic extract did not yield promising results when compared to the control drug metronidazole. When the CHCl₃ and EtOAc fractions of this extract were obtained and tested, an anti-parasitic potential similar to the drug was found (Ibikunle et al., 2011).

The anthelmintic activity depicted by E. uniflora extracts has been attributed to the presence of compounds with proven anthelmintic action, such as flavonoids, saponins, tannins and triterpenes. Different extracts of E. uniflora inhibited the hatchability of eggs of gastrointestinal sheep nematodes, with a percentage of inhibition ranging from 14.56 to 99.75%. The hydroalcoholic extracts were the most promising compared to the aqueous ones. The chemical composition was analyzed using qualitative phytochemical protocols to observe compounds with proven anthelmintic action, such as flavonoids, saponins, tannins and triterpenes. Thus, the results of study revealed that E. uniflora extracts were promising anthelmintic candidates (Castro et al., 2019).

4.4 Antiviral activity

Viruses are unicellular microorganisms that infect cells for survival, triggering an immunological response. For instance, the human immunodeficiency virus (HIV) causes immune system impairment by causing the death of cells involved in the defense mechanism. In this example, the continuous use of retroviral therapy is necessary to reduce the viral load. Notably, some plants can inhibit or reduce viral replication through different mechanisms (Mukhtar et al., 2008) which is the case of E. uniflora.

For instance, when tested against the Dengue Virus Serotype 2 in culture human cells (Huh7-it), E. uniflora leaf extract showed antiviral activity with an IC₅₀ of 19.83 µg.mL⁻¹ (Dewi et al., 2019). It has been described that some flavonoids can inhibit the protease enzymes involved in viral replication (Qamar et al., 2017). For HIV treatment, the use of antiretroviral therapy presents efficacy but causes some side effects associated with the need for continuous administration. Notably, the methanolic and water extracts from E. uniflora leaves showed a replication inhibition at 100 ug.mL⁻¹ (94%) against HIV-1, promoting the inhibition of the protease required in replication and viral action (Kawahata et al., 1996).

Some viruses have long latency periods, therefore they activate during some immunocompromising condition. The Epstein-Barr virus (EBV) is one such virus, causing a condition known as herpes and mononucleosis. Its viral mechanism is associated with the presence of DNA polymerase enzyme that favors replication. The tannins isolated from E. uniflora leaf extract, Eugeniflorin D1 and D2, were found to have inhibitory effect against this virus at concentrations of 3.0 and 3.5 μM, respectively; the mechanism by which they conferred this effect was by blocking the DNA polymerase enzyme (Lee et al., 2000).

4.5 Anti-inflammatory effect

The anti-inflammatory capacity of E. uniflora extract has shown promising results as demonstrated by Bello et al. (2020) who used the ethanolic extract of the leaves in a model of acute formalin-induced inflammation in rats. In this study, the effects of E. uniflora at doses of 50, 100 and 200 mg.kg⁻¹ were similar to those of ibuprofen (100 mg.kg⁻¹, p.o), a standard non-steroidal anti-inflammatory drug (NSAID). This demonstrates the high effectiveness of ethanolic extract against inflammatory processes caused by acute injury. Additionally, the ethanolic extract was able to limit the area of secondary damage in the tissue and to accelerate the recovery of the soft tissue after the injury, leading to the reduction of inflammatory cells in the injured hind paw.

In a different experiment, Falcão et al. (2018) treated carrageenan-induced peritonitis in rats with crude extract, aqueous fraction (AqF) or EAF from E. uniflora leaves. They observed anti-inflammatory properties with reduced levels of IL-1β (Interleukin-1 Beta) following treatment with the crude extract in both fractions and at all doses used (50, 100 and 200 mg.kg⁻¹). Remarkably, the aqueous fraction also stood out due to the decrease in TNF-α (Tumor Necrosis Factor-Alpha) levels. In addition, the anti-inflammatory effect of the extracts and their fractions also showed the reduction of lipid peroxidation that occurred during treatment. This implies that the antioxidant effect would be mediated by an intracellular reduction in glutathione (GSH) consumption, which would cause, in part, increase in the anti-inflammatory potential.

Similarly, E. uniflora leaf extracts (aqueous, ethanolic and essential oil) were assessed by Schapoval et al. (1994). Using the carrageenan-induced rat paw edema test, the authors found that the ethanolic extract (300 mg.kg⁻¹, v.o) was more effective than the aqueous one, which was attributed to the presence of volatile or unstable substances that underwent changes during the time necessary for drying and/or extraction even at room temperature. Using a similar protocol of carrageenan-induced hind-paw edema, Sobeh et al. (2019) found that the methanolic extract from E. uniflora leaves decreased the thickness of the edema and reduced leukocyte migration to the peritoneal cavity. Furthermore, it was demonstrated for the first time that the extract inhibits cyclooxygenases (COX-1 and COX-2) and lipoxygenase (LOX) therefore reducing the production of pro-inflammatory mediators. Although the extract showed greater selectivity for COX-2, its LOX inhibitory activity was similar to that of the diclofenac.

Furthermore, Syama et al. (2019) demonstrated that the leaf ethanolic extract showed an important action in cell line RAW264.7 stimulated by lipopolysaccharide (LPS). The study revealed the extract’s ability to inhibit enzymes that regulate inflammation, which was also confirmed by decrease in the expression of COX-2 and nuclear factor-kB (NF-kB). In vivo carrageenan-induced chronic inflammatory studies in male Wistar rats demonstrated that doses of 250 and 500 mg.kg⁻¹ ethanolic extract of leaves decreased the inflammatory response possibly through the inhibition of pro-inflammatory mediators. This could be attributed to the presence of gallic acid and dihydromyricetin that have proven anti-inflammatory property.

There are reports of E. uniflora’s influence in reducing infection induced by Helicobacter pylori, a bacteria that causes gastric ulcerative disorders that in more severe cases can develop into gastric cancer. Monteiro et al. (2019) demonstrated that leaf E. uniflora methanolic extract decreased inflammatory activity through the use of murine macrophage cell culture RAW 264.7 (ATCC TIB-71) stimulated by LPS. The results demonstrated a reduction in IL-6, TNF-α and nitric oxide (NO) levels at all concentrations tested. However, the concentration 100 µg.mL⁻¹ reached the highest inhibition rate of about 83.19, 39.10 and 97.20%, respectively.

The anti-inflammatory potential of E. uniflora purple pulp juice has also been reported in gingival epithelial cells. It acts by attenuating the release of IL-8 (Interleukin-8) in non-stimulated and prostaglandin-LPS-stimulated cells, respectively (Soares 2014). In addition, important constituents present in the juice such as cyanidin-3-glucoside and oxidoselina-1,3,7(11)-trien-8-one were able to reduce CXCL8 mRNA expression by HGF-1 cells and IL-8 release, thereby revealing an anti-inflammatory potential of the volatile compound oxidoseline-1,3,7(11)-trien-8-one, reported for the first time in the literature.

Another study conducted by Schumacher et al. (2015) reported that the anti-inflammatory effects of E. uniflora leaf extract also contributed to improvement in type 1 diabetes mellitus induced in non-obese rats. Parameters such as decreased inflammatory cell infiltration associated with attenuating oxidative stress, increased levels of hepatic GSH and seric insulin indicate that these associated effects could help preserve insulin-producing pancreatic ß cells and inflammatory insult. However, the authors stated that a better explanation regarding the anti-inflammatory signaling process of the immune response is required.

In addition, the anti-inflammatory effects of these leaves were also confirmed using the Cecal Ligation and Puncture model in mice. The results indicated that 150 and 300 mg.kg⁻¹ doses given orally 1 h before surgery and 6 h after the procedure managed to significantly decrease serum levels of TNF-α by 36.6–38.8%, and levels of IL −1β were also inhibited by 32.3–38.5% after 6 h of induction in the false surgical group. Another important product of cell inflammation was the decrease in induced nitric oxide synthase (iNOS) levels by septic mice ileum cells as E. uniflora’s aqueous extract at all doses (75, 150 and 300 mg.kg⁻¹) decreased iNOS levels by 35.2, 33.6 and 75.2%, respectively. Furthermore, COX-2 expression was reduced by 12.7 and 62% after treatment with higher doses (Rattmann et al., 2012).

4.6 Anti-cancer activity

Anti-cancer activity is based on the cytotoxic potential of a molecule. As previously mentioned, E. uniflora extracts do not cause significant toxicity, especially when the organism or cells are healthy. However, tumorous cells present different membrane and metabolic characteristics, proliferating under oxidative stress and impaired antioxidant defense (Fry and Jacob 2006). Then, it is possible that altering redox status may trigger apoptotic cell death in carcinogenic cells. Therefore, several promising results have been described in this field.

Nuñez et al. (2018) demonstrated that the aqueous leaf extract of E. uniflora showed positive results in a human tumorous cell line derived from invasive cervical carcinoma, SiHa (HPV 16-positive). The data demonstrated that all concentrations of E. uniflora extract (0.5–20 mg.mL⁻¹) significantly inhibited the viability of the SiHa cell line in 24 h and 48 h, and this effect was prolonged at the highest doses of 10 mg.mL⁻¹ 72 h after treatment. In addition, the migration of tumor cells significantly reduced after 24 h of treatment to 63.4% and in 48 h to 24.5%, and the capacity of tumor cell adhesion decreased in a dose-dependent manner (5, 10 and 20 mg.mL⁻¹). In this case, cell death due to apoptosis was also observed in tumor cells treated with E. uniflora and mitigation of the migration, adhesion, colony formation and recovery capacities was observed even after treatment withdrawal without altering the normal cells viability.

Promising results of using E. uniflora against carcinogenic activity were found by Ismiyati et al. (2012) in the breast cancer T47D cell line treated with the extract obtained from the leaves. The results demonstrated an antiproliferative effect at 50 µg.mL⁻¹ 48 h and 72 h hours after incubation. At 75 µg.mL⁻¹, the extract decreased cell proliferation during the entire incubation period (24 h, 48 h and 72 h). Additionally, a cytotoxic effect was also observed, which may have contributed to inhibiting the proliferation of T47D cells and inducing apoptosis.

The cancer cell lines HCT-116 (colon), AGP-01 (malignant gastric ascites) and SKMEL-19 (melanoma) were treated with different E. uniflora leaf oils in a study by Figueiredo et al. (2019). Despite the variations between oils, their constituents had common predominance and belonged to the classes of oxygenated sesquiterpenes (20.8–69.0%) and sesquiterpene hydrocarbons (18.0–53.9%). Figueiredo et al. (2019) demonstrated that curzerene, selin-1,3,7(11)-trien-8-one and selin-1,3,7(11)-trien-8-one epoxide were found in oil extract and all stood out as potential anti-cancer agents for lung, colon, stomach and melanoma tumors. Cytotoxic activity was demonstrated by the two types of oil against all tested HCT-116 cell lines. In addition, curzerene showed the most significant activity against melanoma cells (SKMEL-19), induced apoptosis at 5.0 and 10.0 μM compared to vehicle DMSO and exhibited a decrease in cell migration at 5.0 and 10.0 μM after 30 h of treatment.

Another study using MCF-7 cells (breast cancer cell line) demonstrated that E. uniflora essential oil presented an IC₅₀ of 11.20 μg.mL⁻¹ and a selectivity index of 6.82, making it an interesting candidate for further studies in terms of anti-carcinogenic properties. In this study, E. uniflora oil presented more than 80 compounds, the most predominant of which were from the oxygenated sesquiterpenes class known as spathulenol (15.8%), α-copaene (10.96%), muurola-4,10-dien-1-β-ol (9.3%), caryophyllene oxide (8.93%), allo-aromadendrene (5.5%) and nootkatone (5.17%) (Sobeh et al., 2016).

Finally, Ogunwande at al. (2005) used essential oil extracted from fully grown leaves and fruit and found effects on tumor cells of the human prostate (PC-3), liver (Hep G2) and breast (Hs 578 T). The results identified mostly sesquiterpenoid compounds in both oils. In the leaves, the compounds identified were curzerene (19.7%), selina-1,3,7(11)-trien-8-one (17.8%), atractylone (16.9%) and furanodiene (9.6%), and those from the fruits were germacrone (27.5%), selina-1,3,7(11)-trien-8-one (19.2%), curzerene (11.3%) and oxidoselina-1,3,7(11)-trien- 8-one (11.0%). In this case, the volatile oils exhibited an excellent cytotoxic action in relation to the human cell lines of PC-3 (99.36% and 99.55%) and Hep G2 (99.71% and 99.96%), while completely inhibiting the growth of Hs 578 (100%) by percentage of fruit and leaf oil, respectively.

4.7 Neuroprotective effect

Studies evaluating E. uniflora’s neuroprotective potential are still relatively scarce. Da Da Silva et al. (2019) used the ethanol extract of E. uniflora leaves to observe its neuroprotective effect on memory impairment induced by intracerebroventricular injection of streptozotocin (STZ, i.c.v) in rats. The administration of STZ in rats is a model of sporadic Alzheimer's disease widely accepted for inducing impairment in memory and metabolic changes similar to those of patients. Therefore, rats that received leaf E. uniflora extract at doses of 300 and 1000 mg.kg⁻¹ for 30 days alternately, after i.c.v administration of STZ, demonstrated improvement in episodic memory and learning compared to the untreated animals. Additionally, the performance of the animals that received the extracts were better than untreated animals in both doses tested. The neuroprotective effects have been attributed to the antioxidant and anti-inflammatory properties that have also been described in previous studies using other species of the genus Eugenia. In this case, this study was the first to report potential neuroprotective effects of pitangueira leaves in this neurodegenerative disease model.

The antioxidant and anti-acetylcholinesterase actions of red pitanga extract chronically administered at a dose of 200 mg/kg was analyzed in male Swiss mice in a model of depression. Flores et al (2020) observed promising results regarding the prevention of the depressive effect induced by unpredictable chronic stress, provided by the regulation of acetylcholinesterase activity, reducing the production of reactive oxygen species in the prefrontal cortex and hippocampus and avoiding glutathione peroxidase in the hippocampus of treated animals. It is important to emphasize that the administration of the extract of the red pitanga produced neuroprotection similar to the classic antidepressant fluoxetine, which was used as a positive control. Therefore, these findings may suggest a potential role for the E. uniflora in the treatment of depressive disorders.

Neuroprotective effects of red and purple pulp extracts of E. uniflora were observed in models of Alzheimer's and Parkinson's diseases induced by amyloid β peptide fragment 1–42 (aβ_1-42) and MPP+, respectively, in C. elegans. Borges et al. (2015) demonstrated that the effects induced by aβ_1-42 were reduced in animals treated with both extracts. These could reduce the paralysis phenotype in C. elegans. The authors highlighted that purple pitanga extract presented a higher efficacy than red pitanga extract. Additionally, they also favored important genes of oxidative and thermal stress activation such as the transcription factors daf-16 and Nrf2/Skn-1/inhibition pathways. In the neurodegeneration induced by MPP+, both red and purple pitanga extracts could reduce paralysis in C. elegans, but the red pitanga extract presented the highest percentages of reduction compared to the control.

Based on the data presented, E. uniflora is a promising natural product for the development of new drugs aiming at the central nervous system. The presence of anthocyanins, of which cyanidin-3-O-glucoside is the main compound present in pitanga, demonstrates the neuroprotective mechanism against the formation of free radicals and oxidative processes in the body, since these endogenous processes are related to the genesis of several neurodegenerative diseases.

However, a better understanding of the mechanism of action of the majority compounds such as flavonoids, terpenes and phenolic compounds is still needed, therefore, in addition to components for the development of new drugs, they can become a form of complementary treatment through the diet.

4.8 Antioxidant effect of Eugenia uniflora

Exogenous food antioxidants such as polyphenols and carotenoids can help to protect an organism against diseases associated with oxidative stress (Rahimi-Madiseh et al., 2017). Antioxidant activity of the chemical compound (s) or extract (s) vary according to the model used and also according to the ability of the phytochemical (da Cunha et al., 2016). Currently, several studies have investigated the antioxidant capacities of E. uniflora using in vitro and in vivo assays particularly because this potential might be responsible for several biological advantages attributed to E. uniflora.

4.9 E. uniflora effects on metabolism and TGI

E. uniflora leaves are rich in tannins and flavonoids (Auricchio and Bacchi 2003) and several studies have shown that tannin-rich species have been traditionally used for their gastroprotective properties (de Jesus et al., 2012). In fact, Souza and Costa (2017) demonstrated the gastroprotective effect of an aqueous fraction of hydroacetonic leaf extract of E. uniflora against several gastric ulcer models in mice; increase in the gastric mucus and reduced GSH levels were also found.

Gastric mucus is the first line of defense of the gastric mucosa. It is a transparent, viscous, elastic adherent gel made up of water and glycoproteins (Martins et al., 2015). The mucus layer is a physical barrier that adheres with bicarbonate and protects the underlying mucosa from proteolytic digestion (Allen and Flemstrom 2005). Furthermore, it is known that NO is crucial in the defense of the gastric mucosa and is a biological mediator which regulates the secretion of mucus and blood flow (Falcao Hde et al., 2013). Souza and Costa (2018) demonstrated that an inhibition of NO synthase by L-NAME did not reverse the gastroprotection effect of the aqueous fraction of hydroacetonic leaf extract of E. uniflora, suggesting that NO synthesis is not critical to its gastroprotective activity. Thus, it seems that the primary mechanism of the action of leaf extract of E. uniflora is through increased gastric mucus.

In 2008, a hepatoprotective effect of leaves of E. uniflora against lipid peroxidation was demonstrated in vitro. This can be attributed to the antioxidant properties of E. uniflora extract (Kade et al., 2008). Another study conducted by Fiuza et al. (2009) showed a hepatopancreas action of crude ethanol extract and fractions in Oreochromis niloticus L. Recently, Sobeh et al. (2020) showed that the polyphenol-rich fraction from E. uniflora leaves has substantial hepatoprotective activities against acute liver injury in rats due to its antioxidant property.

Furthermore, a hypotensive activity in normotensive rats has been demonstrated following administration of 3 mg.kg⁻¹ of dried leaves of a crude aqueous extract. This effect was associated with a direct vasodilating action in perfused resistance vessels and a slight diuresis at higher doses (120 mg dried leaves.kg⁻¹) (Consolini et al., 1999, Consolini and Sarubbio 2002). Additionally, a cardiovascular activity caused by aqueous crude extract (ACE) of E. uniflora was demonstrated in rats through β-adrenergic mechanisms. In this case, the release of catecholamines and Ca-blocking action might have produced this therapeutic effect (hypotension) and contributed to chronotropic and inotropic effects on the heart (Consolini and Sarubbio 2002).

In relation to metabolism, folk medicine reports the use of hydro-alcoholic extract of E. uniflora leaves to control the levels of triglycerides, very low-density lipoprotein (VLDL) cholesterol and uric acid in Cebus paella, monkeys (Ferro et al., 1988). Furthermore, Arai et al. (1999) showed that extracts from the leaves of E. uniflora had improved effects on postprandial hyperglycemia and hypertriglyceridemia, and these effects can be attributed to the inhibition of sugar and fat decomposition and reduction of glucose absorption. The red variety of E. uniflora fruit demonstrated effects against high levels of glucose, triacylglycerol, cholesterol and LDL cholesterol, as well as visceral fat and weight accumulation (Oliveira et al., 2017).

Additionally, Schumacher et al. (2015) studied the effects of continuous treatment with aqueous extract of dried leaves of E. uniflora in an experimental model of spontaneous type 1 diabetes mice. This treatment was found to reduce the incidence of type 1 diabetes, decreasing inflammatory cell infiltration and the oxidative stress and increasing hepatic GSH levels and serum insulin. This may indicate preservation of insulin-producing pancreatic β cells.

Another interesting effect was observed with respect to α-glucosidase activity. Even at low concentrations, the ethanolic extract of purple E. uniflora leaves inhibited almost 100% the activity of the aforementioned enzyme (IC₅₀ 0.26 µg.mL⁻¹) (Vinholes and Vizzotto 2017). The E. uniflora fruit juice was also found to inhibit α-glucosidase activity in 69.47 ± 2.89% at 1 mg.mL⁻¹. The total phenolic content of the juice was 367.00 ± 11.42 mg GAE.L ⁻¹ and was considered important to the inhibitory activity observed in the study (Siebert et al., 2020).

This inhibitory effect can be associated with the presence of fatty acids and derivatives. Unsaturated fatty acids such as oleic, linoleic, and linolenic acids and their methyl ester forms inhibit α-glucosidase enzyme through competitive mode inhibition (Su et al., 2013). Furthermore, phenolic compounds such as salicylic acid derivatives showed interaction with α-glucosidase enzyme (Chen et al., 2019). Therefore, the inhibitory properties of the ethanolic extract show promising results as the inhibition of this digestive enzyme is used to control type 2 diabetes mellitus.

Furthermore, E. uniflora fruit (red variety) standardized extract has a beneficial effect in rats submitted to metabolic syndrome induced by diet. This extract presented an antihyperglycemic, antihyperlipidemic and a neuroprotective role as it presented antioxidant and antidepressant-like effects (Oliveira et al., 2018). Moreover, liposomes loaded with an ethanolic extract of purple pitanga were found to reduce lipid accumulation induced by high cholesterol levels in C. elegans (Roncato et al., 2019).

It is evident that in vitro and in vivo studies prove that bioactive compounds present in pitanga can positively affect metabolism biomarkers. The mechanism of action against diabetes have been attributed to the phytochemicals and include modulation of carbohydrate metabolism, glucose homeostasis and insulin secretion, reducing oxidative stress, suppressing the formation of advanced glycation end products and protecting / regenerating pancreatic β-cells. Therefore, leaves and the fruits of E. uniflora have been showing therapeutic potential to be used in the treatment of diabetes and its comorbidities.

These results are interesting as they again point out that natural antimicrobial and antioxidant compounds have shown potential application for the production of active packaging and meet the growing demand from consumers, who are increasingly looking for food products with the lowest amount of artificial additives.