2.1. Total Phenolic Compounds Content
Mamey apple (MA), camapu (C), and uxi (U) were submitted for analysis of total phenolic compounds from extractions submitted with different vehicles solvents (water (W) and ethanol (E)) to determine the most efficient extraction solvent of the mentioned compounds. The results showed that the ethanolic extracts (MAE and UE) of the different Amazonian fruits showed a greater quantity of phenolic compounds when compared to the aqueous extracts (MAW and UW) (Figure 1
). The preparation and extraction from this wide range of samples depends mostly on the nature of the sample matrix and the chemical properties of the phenolics, including molecular structure, polarity, concentration, number of aromatic rings, and hydroxyl groups. Conventional solid-liquid using organic solvent extraction is the main method used to extract phenolics. The sample preparation, polarity of the solvent used, the technique employed and temperature are factors that can influence the extraction and contents of these compounds [14
Uxi ethanolic extract (UE) showed the highest phenolic compound content (701.839 mg GAE/100 g), followed by mamey apple (MAE) (556.105 mg GAE/100 g), and camapu (CE) (237.39 mg GAE/100 g). The aqueous extracts of mamey apple (MAW) and uxi (UW) showed significant difference (p < 0.05) when compared to the respective ethanolic extracts. No significant differences (p > 0.05) were observed in the content of phenolic compounds between the aqueous and ethanolic extracts in the camapu sample.
Aqueous and ethanolic extract showed similar fruits yield extracts, being dependent on the moisture percentage of each fruit. Uxi yield was significantly higher (UE-28.82% and UW-29.42%), followed by mamey apple (MAE-14.04% and MAW-13.13%) and camapu (CE-10.50% and CW-11.10%). Considering these results, the levels of phenolic compounds per mass of fruits for the highest yield would be 202.27, 78.08, and 24.92 mg GAE/100 g for uxi, mamey apple, and camapu, respectively.
Péroumal et al. [15
] studied the pulp of six mamey apple accessions and found values for total phenolic content between 90 and 143 mg GAE/100 g. It can be observed that the results obtained for uxi ethanolic extracts in this work are higher than some Amazonian fruits such as araça-boi (87.2 ± 3.0 mg GAE/100 g) and araça (129.1 ± 9.3 mg GAE/100 g), and lower than camu-camu (1797.2 ± 37.7 mg GAE/100 g) reported by Genovese et al. [16
]. Comparing the results of this study with fruits from the Brazilian Cerrado biome, the Amazonian fruits present lower phenolic compounds in relation to sweet passion fruit (245.36 ± 3.70 mg GAE/100 g), soursop (281 ± 5.40 mg GAE/100 g), murici (334.37 ± 9.07 mg GAE/100 g), and marolo (739.37 ± 7.92 mg GAE/100 g) [17
]. According to the classification proposed by Vasco et al. [18
], MAE, uxi extracts were classified as fruits as having with medium phenolic content (100–500 mg GAE/100 g).
2.2. Phytochemical Profile by UPLC-MSE
For the first time, bioactive compounds of Amazonian fruits like mamey apple, camapu, and uxi were elucidated by UPLC-MSE
metabolomic approach. Globally, 293 compounds were tentatively identified from aqueous and ethanolic extracts of these fruits (Table S1
) and relatively quantified taking account all extracts based on ion counting. Table 1
shows the number of identified compounds and their classification into eight chemical classes according to Phenol Explorer database [19
]: phenolic acid, flavonoids, chalcones, coumarins, amino acid related compounds, fatty acid, and terpene related compounds. The main bioactive compounds in the three species were terpenoids (n
= 61; 21%), phenolic acids (n
= 58; 20%), and flavonoids (n
= 53; 18%). Other metabolites were also identified such as other polyphenols (n
= 50; 17%), including lignans, coumarins and tannins, and also other metabolites (n
= 56; 19%) such as amino acid related, alkaloids and polyketides, showing the extraction and LC-MS methods were suitable to characterize different polarities compounds.
The identification of different phenolic compounds in this study makes it relevant, since the presence of these compounds has a range of bioactivities, as already reported in vitro and in vivo studies [20
]. In a recent study, extracts of phenolic compounds from jatobá-do-cerrado can inhibit α-amylase and α-glycosidase after in vitro digestion and modulate the glucose metabolism [21
Mamey apple was characterized by high number of bioactive compounds. Among the identified metabolites, 209 (71%) and 188 (64%) were found in aqueous and ethanolic extracts of mamey apple, respectively. Furthermore, 183 (62%) and 186 (63%) compounds were found in the aqueous extract ethanolic extracts of camapu, respectively. In uxi, 164 (56%) and 165 (56%) compounds were tentatively identified in the aqueous and ethanolic extracts, respectively. Notably, 50 (17%) compounds were found only in mamey apple (e.g., droserone, norlichexanthone, procyanidin C1, 3,4-leucopelargonidin II, and piquerol A). Piquerol A is a sesquiterpene with low stability in nature and has only previously been tested as an insecticide and as an inhibitor of metabolism in cell cultures [22
]. About 43 (15%) were tentatively identified only in camapu (e.g., myristicin, 11-deoxocucurbitacin, synapic acid I, and pseudopurpurin). Medina et al. [23
] reported the presence of sinapic acid in Passiflora edulis Sims
. Furthermore, about 36 (12%) compounds were found exclusively in the uxi extract (e.g., 4-coumaroylchiquimate, ferulic acid I, kaempferol II, jacareubin, 6-deoxyjacareubin, vanylactic acid II, nigakilactone A, and jasmonic acid). Kaempferol is an important flavonol identified in other fruits like hybrid grapes by Rosso et al. [24
These metabolites are antioxidant inhibitors of mutagenic and carcinogenic compounds and are considered neuroprotective agents in neurodegenerative disorders, such as Parkinson′s and Alzheimer′s diseases [25
The acid phenolic profiling was the most abundant subclass of phenolic compounds in all extracts evaluated, as shown in the Table 1
and Table 2
. Aqueous and ethanolic camapu extracts (CW and CE) showed about 24 and 22% of phenolic acids, respectively, followed by mamey apple aqueous (MAW) and ethanolic (MAE) extracts. In addition to phenolic acids, flavonoids showed high number of identification as well as elevated abundance relative.
Terpenes were between the most abundant bioactive classes in all extracts. About 46 and 43 terpenes, representing 22% of identifications, were found in the MAW and MAE extracts, respectively. In UE extracts, 39 (23%) terpenes were tentatively identified too. Terpenoids are important secondary metabolites of plants and are extremely chemically diversified, being estimated at more than 40,000 substances. Its use has been described as flavoring agents, in addition to providing benefits to human health through its antioxidant potential [26
Although the number of identifications between the extracts of the same fruit was not significantly different, the ethanolic extracts showed a greater relative abundance of compounds in relation to the aqueous extracts in all fruits studied (Figure 2
). This fact can be explained by the large variability of the structures of these bioactive compounds and the proportion of organic solvents with water is required for better extraction.
The principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to explain possible difference among extracts (Figure 3
and Figure 4
). First, the PCA biplot represented both loading (metabolites) and scores (extracts). Such parameters distinguished the profile of bioactive compounds among the fruits evaluated in all extracts. Principal components (PC1 and PC2) demonstrated that the fruits extracts have different composition of bioactive compounds, but the use of aqueous and ethanolic extractors did not favor the removal of distinct metabolite profile in the species. The two major principal components (PC1 and PC2) explained more than 72% of the variance pattern (Figure 3
). Discriminatory metabolites, which showed maximum variance (eigenvalues) among extracts, were observed mainly in the PC2, including the following bioactive compounds bryophyllin A II, 1-O
-glucose I, mammeisin II, eriodictiol II, justicidin A, hallactone B II, 7,2′-dihydroxy-4′-methoxy-isoflavanol, eleganin I, 5-hydroxyferulic acid methyl ester II, lancerin II, 6-methoxytaxifolin II, zapoterin, salvinorin A, meconic acid, benzoic acid II, auriculoside, leucocyanidin II, lophophorine, lancerin I, vernodalol, visnagin, sinapic acid II, sinapyl alcohol II, 6-methoxytaxifolin I, isobrucein A, and syringin I.
Then, HCA was applied to observe the similarity/dissimilarity among the abundance of the discriminatory polyphenols (Figure 4
) in order to understand the profile of composition among extracts. The heatmap indicated that metabolites (20) found in uxi extracts showed higher abundance in comparison to other extracts, especially in the ethanol extracts. Mamey also showed a distinguished bioactive profile, with six compounds found exclusively. Among them, sinapoyl alcohol II is an exclusive metabolite with high abundance in the mamey apple extracts. One of the metabolites with highest abundance in these extracts was syringin. This compound was determined and reported from fresh jaboticaba methanolic extract by Wu et al. [28
] and implicated as immunomodulator having an anti-allergic effect [29
2.3. In Vitro Antioxidant Activity
Due to the multifunctional characteristics of the phenolic compounds found in Amazonian fruits, the effectiveness of measuring the antioxidant capacity of a pulp extract is better evaluated when using commonly accepted tests. The ethanolic and aqueous fruits extracts was determined by four different methods with different action mechanism (ABTS, DPPH, FRAP, and ORAC) (Table 3
). According to Barros et al. [14
], a single assay does not accurately account for all of the groups of antioxidant compounds, because of complexity of fruit matrices, and because of these methodologies can suffer interferences.
ABTS assay revealed values ranged between 263.67 ± 23.90 and 1602.7 ± 30 μmol Trolox g−1
. The highest antioxidant capacity was presented in UE samples (1602.7 ± 30.16 μmol Trolox g−1
). Freitas et al. [30
] analyses fresh pulp of uxi and found 51.6 mg TE/100 g for ABTS. In general, the ethanolic extracts of fruits showed the higher antioxidant capacity for this methodology. However, the aqueous extract from camapu fruit was an exception with a value of 432.74 ± 16.17 μmol Trolox g−1
). Schiassi et al. [31
] studied the activity antioxidant of methanolic extracts of araça (10.92 ± 0.11 μmol Trolox g−1
), buriti (6.03 ± 0.00 μmol Trolox g−1
), cagaita (29.32 ± 0.69 μmol Trolox g−1
), yellow mombin (5.55 ± 0.01 μmol Trolox g−1
), and marolo (132.16 ± 1.40 μmol Trolox g−1
) and found values lower than the present study.
Antioxidant capacity was also evaluated by DPPH•
radical scavenging method (Table 3
) and expressed in aqueous and ethanolic extract concentration μmol Trolox g−1
sample. The highest activity was found in MAE (1168.42 μmol Trolox g−1
), followed by CE (705.771 μmol Trolox g−1
) and UE (509.68 μmol Trolox g−1
). According to Huchin et al. [32
], the DPPH method can analyze hydrophilic and lipophilic compounds, and ethanol is one of the most used solvents for bioactive compounds extraction due, because proposes some benefits, such as low toxicity, good extraction produce, it is safe for human consumption and allows the extracts to be used in the food industry [33
Antioxidant activity by FRAP method followed the same pattern of the DPPH analysis, where higher values were found for the ethanolic extracts. It is probable that the antioxidant compounds of the samples detected by FRAP are the same with those evaluated by DPPH. MAE also presented the highest activity (1381.13 µmol ferrous sulphate g−1
) These results was lower than obtained by Barros et al. [14
] for aqueous extracts of achachairu (712.35 ± 6.61 μmol ferrous sulphate g−1
), araça-boi (798.92 ± 1.52 μmol ferrous sulphate g−1
), and bacaba (6567.45 ± 4.25 μmol ferrous sulphate g−1
Regarding to ORAC assay, the results revealed that UE and UW obtained the highest value (15.04 ± 0.84 and 14.33 ± 1.36 μmol Trolox g−1
), followed by CE and CW (11.15 ± 0.42 and 12.30 ± 1.15 μmol Trolox g−1
). No significant differences (p
> 0.05) were observed between the aqueous and ethanolic extracts of uxi and camapu sample. MAE (8.88 ± 0.52 μmol Trolox g−1
) and MAW (5.17 ± 0.56 μmol Trolox g−1
) showed the lowest values (p
> 0.05) between extracts. Santos et al. [34
] reported to Amazonian fruits such as bacaca (195.00 ± 10.00 μmol Trolox g−1
), buriti (83.00 ± 6.00 μmol Trolox g−1
), inajá (26.00 ± 2.00 μmol Trolox g−1
), pupunha (94.00 ± 1.00 μmol Trolox g−1
), and tucumã (64.00 ± 4.00 μmol Trolox g−1
) results higher than camapu, mamey apple and uxi extracts.
2.4. Correlation between Total Phenolic Compounds and Antioxidant Capacity
The results of Pearson correlation coefficients (r
) between total phenolic compounds and antioxidant capacity of aqueous and methanolic extracts suggest that the total phenolic compounds contributed to the in vitro antioxidant capacity of the extracts according to the method used. The aqueous extract showed a good association of total phenolic compounds with ABTS (r
= 0.843) and ORAC (r
= 0.752) for camapu fruit. ORAC assay has been largely applied to the assessment of free radical scavenging capacity of pure antioxidant compounds and antioxidant plant extracts [35
]. The radical cation ABTS [2,29-]azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) is one of the spectrophotometric methods used to measure water-soluble as well as lipid-soluble antioxidants, pure compounds, and food extracts. The pre-formed radical monocation of 2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•1
) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants [36
-glucose, a compound present in CW (Figure 4
), this compound belongs to the sub class of organic compounds known as hydroxycinnamic acid glycosides. Hydroxycinnamic acids are natural antioxidants found in fruits, vegetables, and cereals [37
Regarding to mamey-apple, the ethanolic extract presented positive correlation with total phenolic compounds in relation to DPPH assay (r
= 0.910). The 2,2-diphenylpicrylhydrazyl (DPPH) assay is widely used in plant biochemistry to evaluate their properties for scavenging free radicals. The method is based on the spectrophotometric measurement of DPPH concentration change resulting from the reaction with an antioxidant [38
According to Figure 4
, the acids syrigin I and visnagin were components with high relative abundancy. Visnagin is an antioxidant furanocoumarin derivative and is a furanochromone that is furo [3,2-g]chromen-5-one which is substituted at positions 4 and 7 by methoxy and methyl groups, respectively. Syringic acid is a dimethoxybenzene that is 3,5-dimethyl ether derivative of gallic acid. It has a role as a plant metabolite, a member of benzoic acids, and derives from a gallic acid. Previous reports described that hydroxybenzoic acids itself and its derivatives showed antioxidant properties against different type of free radicals and can prevent or decrease overproduction of reactive species. The main structural feature responsible for the antioxidative and free radical scavenging activity in the case of phenolic derivatives is the phenolic hydroxyl group. Phenols are able to donate the hydrogen atom of the phenolic OH to the free radicals, thus stopping the propagation chain during the oxidation process. This effect is modulated by the ring substituents, so that electron-withdrawing groups increase the bond-dissociation enthalpy, due to the stabilization of the phenol by a polar structure that leaves a positive charge on the OH group [39
In uxi ethanolic extract, some bioactive compounds were identified with great relative abundance, such as auriculoside, that is a flavan glycoside. Most natural flavans are lipid-soluble and prominent in fruit skin or peel and leaf surfaces, and are usually found at higher concentrations in immature fruits compared to mature fruits [42
]. Lancerin a plant metabolite found in UE is a member of xanthones, a C-glycosyl compound and a polyphenol. Among the polyphenols, the xanthones derivatives comprise an important class of the oxygenated heterocycles with a diversity of substitution patterns that have been described for their antioxidant activity, show to act as metal chela-tors, free radical scavengers, as well as inhibitors of lipid peroxidation [43
]. The presence of these compounds may have contributed to the strong correlation (r
= 0.910) found between total phenolic compounds and ORAC assay.
These results demonstrate the importance of do different methods to available the antioxidant activity, above all in complex matrices. The positive correlation between compound phenolics and ORAC, can be explained due to this assay may be considered a more exact method, because it uses a biologically relevant radical source (peroxyl) and allows the measurement of total antioxidant capacity through the combination of the antioxidant capacity of hydrophilic and lipophilic fractions [44
]. According to the methodology used, these results suggest that phenolic compounds may be one of the main factors responsible for the antioxidant capacity of mamey apple, camapu, and uxi.
2.5. NMR Profile
described the identified compounds for each fruit, with the chemical shift and signal multiplicity, as well as coupling constant (J
), and the quantification in mg/g of freeze-dried fruit. Camapu had the greatest number of different compounds, indicating that it has a large variety of nutrients and metabolites, even more than could be extracted and identified. Uxi had the least amount, probably due to the great quantity of lipophilic compounds that could not be extracted, so this result does not mean that there are few compounds in uxi.
Sugars were most abundant compounds in those fruits. Camapu showed the highest amount of sucrose, and mamey apple showed the highest amount of fructose and glucose, both α and β. In the uxi extracts no sugars were found. This result indicates that mamey apple and camapu are sweeter than uxi, and probably would be more accepted by consumers. The sugars present in mamey apple were sucrose (1.50 ± 0.10 mg/g), fructose (0.85 ±0.03 mg/g), α-glucose, and β-glucose (0.49 ± 0.02 and 0.40 ± 0.01 mg/g, respectively). The sugars found in camapu were sucrose (4.09 ± 0.46 mg/g), followed by fructose, α-glucose, and β-glucose (0.11 ± 0.00, 0.04 ± 0.00, and 0.04 ± 0.00 mg/g respectively).
Fruit and vegetable flavor depend upon taste (given by sweetness, sourness, acidity, and astringency) and aroma (concentrations of odor-active volatile compounds). Sweetness is determined by the concentrations of the predominant sugars, which are ranked relative to sucrose (fructose > sucrose > glucose). Sourness or acidity is determined by the predominant organic acids, which are ranked relative to citric acid (citric > malic > tartaric). Some amino acids, such as aspartic and glutamic, may also contribute to sourness. In general, consumer acceptance is related to soluble solids concentration (sugars, organic acids, soluble pectin, some phenolic compounds, and ascorbic acid) or the ratio of soluble solids to titratable acidity [45
It has been shown that mamay apple has ethanol (0.01 ± 0.00 mg/g), which is possibly a fruit sugar fermentation product. It was also found choline (0.01 ± 0.00 mg/g) and organic acids, such as formic acid and shikimic acid (0.01 ± 0.00 and 0.11 ± 0.00 mg/g) in mamey apple. Shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) is natural acid from the plant metabolism and common in berries fruits. It is a precursor for the biosynthesis of primary metabolites such as aromatic amino acids and folic acid, and a great many other aromatic compounds. The benzene ring is formed through the shikimate pathway, and shikimic acid is an extremely essential compound in plants and microbes. Shikimic acid has been found to occur in many tissues of a variety of plants, with a sufficiently high percentage. Moreover, its content and accumulation in different tissues depends on the rate of metabolic processes taking place in them. Shikimic acid is utilized for industrial synthesis of the oseltamivir (antiviral), zeylenone (employed as a preparation for chemotherapy of cancer), and monopalmityloxy shikimic acid (anticoagulant activity). In addition, shikimic acid derivatives represent a great interest for agriculture because many of them are used as herbicides and antibacterial agents [46
Choline is a natural amine that can be synthesized in human body, but this production usually is not enough to meet human needs in men and postmenopausal women. Some important functions of choline are it is a part of the neurotransmitter acetylcholine; it is a part of the predominant phospholipids in membranes; it forms betaine, which is an important osmolyte in the kidney glomerulus and helps with the reabsorption of water from the kidney tubule. Eggs and liver are the main sources of choline, but many other foods contain significant amounts of choline and esters of choline [47
Ethanol (0.02 ± 0.00 mg/g) was also present in camapu, indicating fermentation. The organic acids found in camapu were lactic acid, acetic acid and formic acid (0.01 ± 0.00, 0.01 ± 0.00, and 0.01 ± 0.00). Besides those organic acids, it was found γ-amino butyric acid (GABA) in camapu (0.02 ± 0.00 mg/g). GABA is a four-carbon nonprotein amino acid and is widespread in bacteria, animals, and plants. GABA is naturally present in small quantities in plants, and is produced in response to anaerobic conditions, γ
-radiation, low pH, low or high temperatures, and darkness, and by mechanical manipulation. Some functions of GABA in plants are: regulation of cytosolic pH, protection against oxidative stress, defense against insects, and the regulation of pollen tube growth and guidance [48
]. In vertebrates is a neurotransmitter that is deficient in the brain of people with Alzheimer disease [49
]. Camapu has been shown to be effective in delaying the development of this disease and in its treatment, by increasing the proliferation of neural stem cells in vivo [50
] and also shown an anxiolytic effect [51
] which the authors suppose is due to the presence of GABA agonists. Several amino acids such as aspartic acid, alanine, and valine (0.03 ± 0.00, 0.02 ± 0.00, and 0.01 ± 0.08 mg/g) and choline (0.01 ± 0.00 mg/g) were also present in camapu in sufficient quantities to be identified.
For uxi, the compound with in the highest amount was linoleic acid (1.79 ± 0.10 mg/g), which is the main fatty acid of ω-6 group. This result shows that there are fatty acids that are sufficient to appear in a spectrum of fingerprints, even though other lipids may not have been completely extracted from the sample. There is probably a low amount of sugar in uxi fruit because there are high amounts of lipids and usually sugar and lipids in the pulp of fruits are inversely proportional. However, it was found ethanol (0.15 ± 0.01 mg/g), indicating that some sugar that was present before it was fermented. It was also found enough amount of acetic acid (0.15 ± 0.00 mg/g) valine and alanine amino acids (0.05 ± 0.00 and 0.04 ± 0.00 mg/g, respectively).