Characterization of Bioactive Compounds in Northern Amazon Fruits

Fruits and vegetables are highly appreciated because they are constituted by active phytochemicals with functional properties for the organism acting with modulating pharmacological effect. Given the pharmacological properties of this type of food, in this work were studied the concentrations of vitamin C, total carotenoids and reducing and non-reducing sugars of nine fruits developed in the northern Amazon region: Abiu, Acerola , Araçá , Bacuparí , Biribá , Camu-camu , Fruta-do-conde , Graviola and Tapereba . The concentration of vitamin C, the highest concentration in the shell of Camu-camu 2521.51 mg 100 g -1 and for acerola with 1731.4 mg 100 g -1 stand out. The highest concentrations of total carotenoids were also found for the Camu-camu , with concentrations of 0.67 mg 100 g -1 the shell of Camu-camu and 0.57 mg 100 g -1 for the pulp. The concentrations of sugars are higher for the pulps, with the highest concentrations for the pulp of the Fruta-do-conde with 16.31 g 100 g -1 followed by the pulp of the Graviola , both of the Annonaceae family with a concentration of 15.61 g 100 g -1 . The different bioactive molecules were correlated for the different parts of the fruit, by means of multivariate analysis techniques (PCA and HCA), where 90.1% of the cases were explained for the pulps, 65.4% for the shell of the fruits and finally the 88.5% of the cases for the seeds. Given the results obtained in this work, these fruits can be used for the preparation of foods with functional interest.


Introduction
The antioxidant capacity of fruits varies as to its composition in phenolic compounds such as carotenoids and flavonoids as well as the concentrations in vitamins E and ascorbic acid (Saura-Calixto & Goñi, 2006). The phenolic compounds that exist in plant sources are divided into flavonoids and non-flavonoids, while the anthocyanins and carotenoids are within the flavonoids and the color of antioxidants is related to the molecular structure (Rodríguez-Amaya, 2004). Foods rich in these active molecules, have aroused increasing interest among consumers, especially in the European and North American continent, where there is 92% of consumption of this type of products (Sahota, 2015). The bioactive compounds are functional ingredients for foods that provide health benefits, as well as influence physiological processes that reduce the risk of chronic diseases, including antioxidants, carotenoids and vitamins (Jiménez-Colmenero, 2013;Cantillano et al., 2013). (2000), due to the antioxidant capacity of this acid, it is one of the main vitamins for human nutrition, due to its main active molecule, L-ascorbic acid.
Another group of biomolecules of interest in the quality of the fruit are the sugars, since they are related to the flavor of the same, since as the fruit matures, the amount of soluble organic acids decreases and the quantity of soluble sugars increases, acquiring the same sweet taste. Of the simple sugars present in fruits, fructose and glycose stand out, with lower concentrations of sucrose and sorbitol (Barreiros, Bossolan, & Trinidade, 2005).
The objective of this work was to quantify the content of vitamin C, total carotenoids and reducing and non-reducing sugars in pulps, peels and seeds of fruits developed in the northern Amazon and correlate the different biomolecules by chemometric techniques of multivariate analysis.

Collection and Preparation of Samples
Samples of the different species studied in this work were collected in several points of the State of Roraima (Brazil), as well as in the markets of the city of Boa Vista and directly with producers who owned the fruits, in order to prepare a representative composite sample for each of the fruits, their botanical and common names being shown in Table 1. The samples were taken to the laboratory of Environmental Chemistry of the Federal University of Roraima (Brazil), where they were selected those that had an excellent state of conservation, washed previously with distilled water, then with 1% sodium hypochlorite solution and again with distilled water.
After separating the material in pulp, shell and seed, they were taken to the laboratory of the Agronomic Research Center (NUPAGRI), at the Agricultural Sciences Center, Cauamé Campus, UFRR, where they were lyophilized in LÍOTOP liquefaction model L101 for 48 hours until drying the material. Subsequently, the material was dried, milled with LABOR model SP31 knife mill and placed in hermetically sealed bags and stored away from the light until the analyzes were carried out.

Quantification of Vitamin C by Reduction of Cupric Ion
Weighed 2.0 g of lyophilized material and transferred to an Erlenmeyer flask with 10 mL of 5% metaphosphoric acid solution and 10 mL of 0.05 M sulfuric acid. Posteriorly, the samples were agitated for 30 minutes and after agitation, 10 g were transferred to a 50 mL volumetric flask and were completed with one volume of water. From there, an aliquot of 10 mL was taken and 5 mL of chloroform was added to the flask and stirred for 1 minute. Then they were left at rest to separate the layers and 5 mL of the aqueous (limpid) layer was pipetted and taken to a separating flask, where 1 mL of the buffer solution and 5 mL of the complexing solution were added. Stir later for 90 seconds and let the layers separate. Remove 3 mL of the top part (isoamyl alcohol) and transfer to a 25 mL Erlenmeyer flask. Add 0.5 mL of isoamyl alcohol and shake gently. Prepare the blank, pipetting 5 mL of solution A (pipette 2 mL of 5% metaphosphoric acid solution, transferred to a 100 mL volumetric flask with distilled water) directly into the reaction tube and then with the sample, making the readings at 545 nm. The calibration curve was prepared by diluting aliquots of the standard solution of 0.5: 1.0; 1.5; 2.0 and 2.5 mL of the standard solution 20 μg mL -1 (This solution is prepared from the dilution of a concentration of ascorbic acid PA of concentration 1 mg mL -1 for a 100 mL flask and add 2 mL of the 5% metaphosphoric acid solution completing the volume with distilled water) corresponding to 10, 20, 30, 40 e 50 μg of ascorbic acid and completing up to 5 mL of solution A. Subsequently, 1 mL of the buffer solution and 5 mL of the complexing solution. Shake the solution vigorously and let the layers separate. From the upper layer, 3 mL of the upper part is removed and transferred to a 25 mL Erlenmeyer flask and 0.5 mL of isoamyl alcohol is added, shake and read at 544 nm in UV-visivel molecular absorption spectrophotometer model SHIMADZU UV -1800 (Badolato et al., 1996;Contreras-Guzmán, 1984).

Quantification of Sugars
Using the methodology described in IAL (2008), certain carbohydrates are not present in different fruit. This section is determined by the reducing agents and not reducers. The first ones are determined by a Fehling reaction, using the Lane-Aynon method, onde or aldehyde or ketonic free group in position C1, or sugar considered a redutor, thus reducing indicators, as the complexes of (Cu 2+ ) to cuprous form (Cu + ). Or agent in these reactions is an open chain form of aldose or ketosis. On the other hand, non-reducing sugars are not determined, but based on reductive sugar (copper sugar sulphate) in the alkaline (Fehling's solution), forming a precipitate of cupric oxide. After hydrolysis acid two non-reducing disaccharides. This method will determine the theory of non-reducing glycides (Macedo, 2005).

Determination of Total Carotenoids
The determination of total carotenoids was also performed by UV-visible molecular spectrophotometry, in a spectrophotometer model SHIMADZU UV-1800 by the technique described by Lichtenthaler and Buschmann (2001) modified, where 1 g of lyophilized material was weighed to which was added 18 mL of acetone, the carotenoids being extracted by shaking for 20 minutes in the absence of light. The samples are filtered and the absorbance readings are carried out at concentrations of 661 nm, 644 nm and 470 nm respectively, to calculate the carotenoid concentration by means of equations 1-3.

Statistic Analysis
Correlations between the amounts of the different minerals in the seeds of the fruit were evaluated using the statistical program INFOSTAT Rienzo et al. (2016) for significance levels of 5%, 1% and 0.1% respectively, as well as the principal component analyzes (PCA) and Hierarchical component analysis (HCA).

Result
Tables 2-4 show the results of ascorbic acid, total carotenoids and sugars (reducing and non-reducing) for the pulp, skin and seeds respectively.

Determ
In  Vol. 11, No. 9;2019 is 1.18 mg 100 g -1 , slightly lower than the value found in this study. Taperebá pulp, the concentration of total sugars is low compared to other fruits, but with values close to those determined by Mattietto et al. (2010), who found sugar concentrations of 4.54 g and 100 g -1 .
In Table 3, the results of the concentrations of the bioactive molecules for the shells of the fruits studied are presented. In comparison, the values of vitamin C between the pulp and the shell of the fruits, it is found that these are superior to those of the pulps, being generally a discarded part of the fruit. For the shell, the lowest vitamin C values were found in the taperebá shell with only 34.17 mg 100 g -1 and the highest values again for the shell of acerola and camu-camu. Correa et al. (2011), study the pulp and shell of camu-camu in different stages of maturity, where they also determine concentrations of vitamin C in the shell above the pulp, concentrating vitamin C in the shell as it matures. The fruit, being the determined values of vitamin C of 2792.82-2496.23 mg 100 g -1 of camu-camu shell, these values being within those found in this work. Carotenoids already have higher concentrations than pulp, with the lowest value for taperebá shell 0.041 mg 100 g -1 and the highest values for camu-camu shell 0.67 mg 100 g -1 and acerola 0.47 mg 100 g -1 . The concentration of these compounds is greater in the shell of fruits since these compounds have as a mission to protect plant cells from oxidation and free radicals, in addition to promoting the formation of antibodies that act specifically against substances or elements strangers that can affect the organism (Palencia, 2010).
The concentration of sugars in the shell of the different fruits is lower than that found in the pulps, with the lowest concentrations in the araçá shell with 1.07 g 100 g -1 and 10.37 g 100 g -1 the highest concentration, for the shell of the abiu. The sugars together with the acidity influence the sensory quality of the fruit, being found in ripe fruits, simple sugars such as sucrose, glucose and fructose acting as primary source of energy (Miller et al., 1986).
In Table 4, the results for the seeds are presented, where the concentration of vitamin C in the seeds of the fruits studied was not detected in the taperebá seed and in others the values were extremely low as in the case of the biribá with 7.13 mg 100 g -1 and the graviola with 7.84 mg 100 g -1 being again, the highest values of vitamin C for the camu-camu and acerola seeds. The vitamin C content in the camu-camu seed was studied by Neves et al. (2015), in a flour obtained with the shell residue together with the seed where the concentration of ascorbic acid was 9004 mg 100 g -1 , values higher than those of the husk and isolated pulp, as it happens in this work, where the concentration is higher than for the other parts studied. The content of vitamin C decreases in fruits as they mature as well as with storage, since the acid ascorbic acid oxidase (ascorbinase) acts directly or by the action of oxidizing enzymes such as peroxidase (Poliniati, 2010). For acerola seeds, the value determined in this work is close to that determined by Aguiar et al. (2010), which obtain a concentration value of ascorbic acid of 66 mg 100 g -1 .
The concentration of carotenoids found in the seeds of the fruits studied, these are found in low concentrations compared to other parts of the fruit studied, with the lowest values for the seed of the Abiu with 0.012 mg 100 g -1 and the highest value for the seeds of biribá and camu-camu with 0.094 mg 100 g -1 . Given their characteristic of being soluble in vegetable oils, they give pigmentation to the different vegetable oils concentrated mainly in the seeds. The main carotenes found in vegetable oils are -carotene, -carotene and phytoene (Ferrari, 2001). According to Basu et al. (2001), carotenoids play an important role in the cellular protection against lipid peroxidation, preventing degenerative diseases such as cancer, heart disease or reduction in the creation of cataracts and strengthening of the immune system.
Finally, the concentration of total sugars in the seeds varies between 7.15 g 100 g -1 for the Araçá seed, 18.25 g 100 g -1 for the camu-camu seed. These fruit seeds are richer in reducing sugars than in non-reducing sugars (Table 4). Within these reducing sugars are glucose, fructose, maltose and aldose that can be reduced in the presence of transition elements such as copper or iron (Demiate et al., 2002).

Analysis of Main Components (PCA) in Different Parts of the Fruits
In the blipot (Figure 1), the results of the principal component analysis (PCA) for the concentration of the bioactive molecules in the different pulps studied are presented, explaining 90.1% of the original variability of the data retained in these components.
The arrangement of the sequence in Figure 1, shows that the systems can be grouped into two sets, the first major component (CP1), contributed with 61.9% of the total variance explained, however most of the variables that were strongly affected contributed positively to CP1 carotenoids and vitamin C, and inverse with reducing and non-reducing sugars. These results indicate that CP1 allowed to distinguish the fruits that are associated with the pulp of camu-camu and acerola which are strongly associated. The second main component (CP2) explained 26.2% of the total data, appearing in the values of reducing and non-reducing sugars. The analysis of this component also showed that this attribute projects negatively on vitamin C and carotenoids. graviola, fruta-do-conde, biribá, abiu and bacupari pulps were associated.
In the blipot (Figure 2), the results of the analysis of the main components (PCA) for the bioactive molecules of the shell of the fruits studied are explained, explaining the 95.6% of the original variability of the data retained in these components.
The arrangement of the sequence in Figure 2 shows that the systems can be grouped into two sets, the first major component (CP1), contributed 65.4% of the total variance explained, however most of the variables that were strongly affected contributed positively to CP1 the vitamin C and carotenoids and inverse with the reducing and non-reducing sugars as happens with the pulps studied. These results indicate that CP1 allowed distinguishing that camu-camu and acerola are strongly associated. The second main component (CP2) explained 30.2% of the total data, appearing in this case the reducing and non-reducing sugars. The analysis of this component also showed that this attribute projects negatively on vitamin C and carotenoids, and the bioactive molecules in the barks of biribá, fruta-do-conde, graviola, abiu and bacuparí showed to be associated.
In the blipot (Figure 3), the results of the main component analyzes (PCA) for the bioactive molecules of the seeds of the fruits studied are represented, accounting for 88.5% of the original variability of the data retained in these components. The arrangement of the sequence in Figure 3 shows that the systems can be grouped into two sets, the first major component (CP1), contributed with 73.2% of the total variance explained, however most of the variables that were strongly affected contributed positively to CP1 vitamin C and sugars and to a lesser extent carotenoids. These results indicate that CP1 allowed distinguishing that araçá and camu-camu and acerola are strongly associated. The second main component (CP2) explained 15.3% of the total data, appearing in this case associated with the remaining fruit seeds.

Hierarchical Analysis of Components (HCA) in Diferent Parts of the Fruits
In Figure 4, the hierarchical component analysis (PCA) for fruit pulps is presented. The bioactive molecules in the pulps of the fruits studied, the trends observed through the analysis of PCA main components, were observed through the HCA, mainly observing four large groupings: one of them formed by the association of graviola with the fruta-do-conde and biribá with smaller Euclidean distance, belonging them the same family. Another strong association is the bacuparí pulp with abiu. Taperebá with araçá and acerola with camu-camu. All fruit pulps not counting the abiu and acerola are associated with the euclidean distance of 10.97, grouping with the other two fruit pulps with euclidean distance of 21.93 In Figure 5 the HCA analysis for the skin of the different fruits is shell. As it ocorre in the pulp of the fruits studied, in the shell there are four groups. On the one hand, the camu-camu with the acerola that are strongly associated and only joins with the rest of fruits at an elevated Euclidean distance of 22.99. On the other hand, we have an association between the shell of the biribá with the bacuparí that are associated in turn to a Euclidean distance with the abiu, graviola with the fruta-do-conde are associated and the other grouping is between the taperebá and araçá. The dendrogram presented for the seeds ( Figure 6) presents atypical behavior if we compare it with the other parts of the fruit. Fruits that appear strongly associated (less Euclidean distance) are the graviola with bacuparí and practically at the same distance the fruta-do-conde with the biribá that in turn the bioactive molecules in this group of fruits are going to be associated abiu at a slightly higher Euclidean distance.

Conclusions
The results obtained show that the fruits studied have a good source of bioactive compounds, especially acerola and camu-camu, where the concentration of ascorbic acid is especially high in the pulp and in the shell. These fruits are at the same time the source of carotenoids and sugars, with the highest concentrations for abiu, fruta-do-conde and graviola. These fruits, due to the concentrations of these compounds, present socioeconomic potential for the region where they are found, and can be consumed in natura or industrialized in the form of foods with high functional potential.