Bioactive compounds in fruits from different regions of Brazil Compuestos bioactivos en frutas de diferentes regiones del Brasil

Este trabajo fue recibido el 09 de noviembre de 2018. Aceptado con modificaciones: 28 de mayo de 2019. Aceptado para ser publicado: 13 de agosto de 2019 ABSTRACT It is important to determine the chemical composition of fruits in order to establish their potential health benefits for human nutrition and thus stimulate their cultivation and consumption. Therefore, the objective of this study was to determine the physical and chemical parameters, vitamin C content, phenolic compounds, flavonoids and carotenoids, fatty acid profile and antioxidant activity of fruits from different regions of Brazil. We observed that the different regions and fruits studied presented very distinct characteristics. For example, the cupuaçu pit is rich in unsaturated fatty acids, while the genipap and java plum presented high flavonoid content and the butia had a high concentration of carotenoids. Guava was the fruit with the highest antioxidant activity through the ABTS radical, while the yellow mombim presented the best response when the activity was determined by FRAP.


INTRODUCTION
The search for healthy food sources that provide health benefits is a topic of growing interest, motivating diets based on fruits and vegetables, which are directly associated with reducing the incidence of chronic and degenerative diseases. Plants present considerable amounts of micronutrients, such as minerals, fibers, bioactive compounds, and a balanced profile of fatty acids 1 . Brazil has a vast plant biodiversity, and is the third largest fruit producer in the world. Many fruits from Brazil have peculiar sensorial characteristics and nutritional and economic potential, influencing their consumption for national and international markets 2 .
Fruit pulps and related co-products have become popular and are now used in supplements, dietary additives and pharmaceuticals, with the aim of controlling and / or preventing diseases 3 . However, research on the composition of bio compounds, which are related to the functional properties of fruits grown in different regions of Brazil, is still scarce. Thus, it is important to study a number of fruit species and focus on the chemical characterization and number of bioactive compounds. This information would provide knowledge that could be incorporated in agribusiness, leading to more usage of raw materials hitherto little used in the food and pharmaceutical industries 4 . Among the compounds with functional properties, substances with antioxidant activity receive special attention for protecting the human body against oxidative stress, and preventing several chronic degenerative disorders 5 . Fruits have a variety of antioxidant compounds, such as vitamin C, phenolic compounds and carotenoids. Vitamin C refers to all compounds which exhibit biological activity equivalent to L-ascorbic acid. It is one of the most important water-soluble vitamins for human health, known for its antioxidant activity and is also involved in many biochemical functions. It is often used as an additive by the food industry to avoid the oxidation of food products 6 . Vitamin C captures reactive oxygen species forming free radicals, such as semi dehydroascorbic acid or ascorbate radical, from the loss of one electron 7 .
Phenolic compounds are classified into groups according to their chemical structure, such as flavonoids, phenolic acids and tannins. The antioxidant activity and anticancer and antimutagenic effects of phenolic compounds have been widely reported 1,8 . Their antioxidant action is attributed to the reduction in power of the aromatic hydroxyl group in addition to chelating transition metals 3,9 . Carotenoids, which are liposoluble pigments, can act as natural antioxidants, protecting the cells against reactive oxygen species and free radicals, acting mainly via energy absorption that arises from the system of conjugated double bonds present in their structure 10 .
As with bioactive compounds, the profile of fatty acids present in the oils of the seeds of some fruits influences functional properties, since a balanced composition of saturated and unsaturated fatty acids confers high nutritional values 1,11 . Unsaturated fatty acids such as oleic acid (ω-9), linoleic (ω-6) and linolenic acid (ω-3) are considered indispensable components for the structure, development and function of cells, as they are directly related to a decrease in the risk of numerous diseases, especially cardiovascular, cancer and metabolic syndrome 11 .
In this context, it is important to determine the chemical composition of fruits in order to establish their health benefits for human nutrition and thus to stimulate their cultivation and consumption. Therefore, the objective of this study was to determine some physical and chemical parameters, the vitamin C content, phenolic compounds, flavonoids and carotenoids, the fatty acid profile and the antioxidant activity of fruits from different regions of Brazil. Twenty-six mature fruits were purchased from local  companies from different regions of Brazil from January  to June of 2018, which are presented in table 1. The fruits were selected with respect to health, physical integrity, color uniformity and degree of maturity. After being sanitized, the seeds and husks were separated and the parts were crushed and stored in a freezer at -80°C until the analysis time.

Physical chemical determinations
Aluminum capsules were weighed after being kept in an oven at 105 °C for one hour. Next approximately 5 g of sample per capsule was weighed and placed in an oven (105 °C) for 4 hours, the results expressed in % of humidity 12 .
The total soluble solids content was obtained by measuring the refractive index in an Abbé Analytik Jena refractometer with automatic temperature correction to 20 °C, calibrated with distilled water. Results were expressed in °Brix in accordance with the methodology of the AOAC 12 .
Acidity was determined by titrating one gram of sample with sodium hydroxide solution, in accordance with the AOAC 12 . The results were expressed as mg 100 g -1 citric acid. The pH was determined in a bench pH meter in accordance with the methodology described by the AOAC 12 .

Bioactive Compounds
The total content of phenolic compounds was determined following the methodology of Singleton and Rossi 13 . Absorbance was measured at 765 nm in a spectrophotometer, with white methanol PA and results were expressed in mg equivalent of gallic acid per 100 g of dry matter (mg EAG.100 g -1 ).
Aliquots of 2 mL of fruit pulps at 20 μg mL -1 were added with 1mL of 2.5% (w/v) aluminum chloride solution. The absorbance reading was taken after 30 minutes at 415 nm in a spectrophotometer, and the results were expressed in milligram equivalents of quercetin per 100 g of dry matter (mg EQ 100 g -1 ).
The total carotenoid content was determined according to the methodology described by Rodriguez-Amaya 14 . The results expressed in milligrams per 100 g of dry matter. Vitamin C content was determined by titration following the method described by AOAC 12 and expressed as mg of ascorbic acid in 100 ml -1 of juice.

Fatty acids
Fatty acid analysis was performed only on fruits that contained kernels (Butia, Pequi, Bacuri, Cupuaçu and Pupunha). Using the methodology of Hartman and Lago 17 , an aliquot which contains fatty acid methyl esters was collected in a vial and injected into a Perkin Elmer Clarus gaseous gas chromatograph equipped with a FID detector and Carbowax ID 20 M column of 0.25 μm and dimensions 30 m x 0.25 mm, coated with polyethylene glycol. Nitrogen was used as the entrainment gas at 1.5 mL.min -1 . Fatty acids were identified by comparison with the retention times of methyl esters (Sigma Chemicals Co., St. Louis, USA). Results were expressed as relative percentage of fatty acids.

RESULTS
Considering the moisture content of the fruits (Table  2), it was found that pequi, assai, pupunha, jabota, custard apple and sapoti had moisture contents below 80%. Souza et al. 18 reported moisture between 80.16 and 93.48% for fruit pulps of the Brazilian cerrado biome, whereas in the present study this variation was from 24.08 to 92.19%. The fruits showing these extremes were jatoba and biri-biri, respectively. The jatoba, which has a pulp with a farinaceous aspect, was the only fruit with humidity below 50%, so it can be suggested that the jatoba fruit is suitable for the elaboration of flour and other solid foods.
Among the fruit examined, pequi, caja-manga, assai, guava, bush banana, dragon fruit, passion fruit, genipap, biri- biri, West Indian gherkin, yellow mombim, Seville orange, peach and African horned cucumber presented soluble solids content between 0.6 at 5.0°Brix; capuaçu, java plum, butia, persimmon, jatoba, cajarana and star fruit presented total soluble solids content between 6.0 and 10°Brix; and only the custard apple had content above 10°Brix (15°Brix). Souza et al. 18 evaluated fruits of the cerrado biome and found a variation of 1.0 to 13.3°Brix. Jatoba was the only fruit that presented a high sugar content and low moisture content, which suggests that the fruit lasts longer. However, other factors may be related, such as fruits harvested at less advanced stages of maturation that are exposed to water loss for a longer time; i.e. they perspire more, so the concentration of the cellular juice, and consequently the concentration of sugars, occurs.
Acidity is an important parameter in the quality of products of plant origin, besides being related to the conservation of the products for a longer period of time 14 . Among the analyzed fruits, pequi, caja-manga, assai, pupunha, guava, dragon fruit, passion fruit, genipap, West Indian gherkin, Seville orange, peach, cupuaçu, java plum, butia, persimmon and sapoti presented an acidity between 0.08 and 0.96% in citric acid. On the other hand, jatoba, bush banana and yellow mombim had an acidity between 1.18 and 1.56% in citric acid. A relationship between acidity and sugar content was not observed in the fruits analyzed. A high value in the ratio of total soluble solids and titratable acidity indicates an excellent combination of sugar and acid that correlates with the mild taste of the fruits; while low values correlate with higher acidity and consequently with less desirable flavor of the fruits.  The fruits studied presented pH values ranging from 2.88 to 5.48, with caja and jatoba representing these extremes, respectively. Most fruits and vegetables fall into the group of acidic foods (pH 4.0-4.5) or very acidic foods (pH <4.0), which restricts the growth of pathogenic microorganisms.
A significant differentiation of vitamin C intake was made (Table 3). Values ranged from 0.55 to 3.70 mg.100g -1 in sapoti, pequi, dragon fruit, pupunha, bush banana and Seville orange fruits in the states of Ceara, Goias, Mato Grosso do Sul, Para and Rio Grande do Sul, respectively. The effects of vitamin C on fruits may vary due to the variety and degree of maturity, as well as the region and cultivation practices, which are influenced by climatic conditions and soil composition 19 . Souza et al. 18 found vitamin C content in Table 3. Total content of phenolic compounds (mg E AG.100g -1 ), flavonoids (mg EQ.100g -1 ) and vitamin C (mg ascorbic acid.100g -1 ) ± standard deviation of fruits from different regions of Brazil. Cerrado fruits between 21.83 and 59.05 mg.100g -1 Among the studied fruits, the butia and the pupunha were those that presented the highest content of carotenoids, whereas the lower contents were found in the persimmon and jatoba fruits (Figure 1).
Among the fruits analyzed, those with the highest potential antioxidants (Table 4) were guava, java plum and butia (Rio Grande do Sul) and biri biri (Bahia), for both methods tested. High values were also observed for custard apple, genipap and Seville orange by the ABTS method, and for the bush banana and yellow mombim by FRAP. The fruit with the lowest antioxidant potential by both methods was sapoti, and no reduction was observed by the FRAP method. Between FRAP and ABTS, the antioxidant activities were not similar, and it is possible to observe that the compounds that act to reduce iron to the FRAP method are not the same ones that act in the ABTS radical inhibition and vice versa. Similar results were observed in the work of Almeida et al. 21 when antioxidant activity was measured via the DPPH and FRAP methods.
According to table 5, it can be observed that of the fruit pits analyzed, cupuaçu presented the highest content of unsaturated fatty acids, of which oleic acid was the main fatty acid found. This fatty acid belongs to the ω9 family and helps in the decrease of low-density lipoprotein in plasma and in the reduction of risk of cardiovascular diseases; followed by linoleic acid, which is not synthesized by the human organism and must be obtained from diet. Deficiency can result in squamous skin lesions, growth retardation and thrombocytopenia 25 .
The cupuaçu pit also presented a high relative percentage of gadoleic acid (11.50%). In relation to saturated fatty acids, cupuaçu pit oil presented only 9.38%. The oil of the pequi pit also stood out in relation to unsaturated fatty acids, of which oleic acid presented 52.76% and linoleic acid 8.52%.

DISCUSSION
Moisture is one of the most important factors affecting food, as it has a direct effect on maintaining quality. Moisture is the total amount of water contained in a food, i.e. water that is closely bound to the constituent molecules of the product, cannot be removed or used for any kind of reaction (water combined), and free water, which is available for physical (evaporation), chemical (darkening) and microbiological reactions, becoming the main cause of its deterioration. High moisture content contributes to a lower conservation  The total soluble solids content is used as an indirect measure of the sugar content. Although other substances are also dissolved in the vacuolar sap, sugars are the most representative, accounting for 85% to 90% of total soluble solids. Soluble solids content is an important characteristic of products that are consumed fresh, as consumers prefer sweeter fruits 26 .
pH values below 4.5 are desirable to prevent the proliferation of microorganisms. There was no direct relationship between high acidity and the lowest pH value in the fruits. The relationship between pH and the dissociation of organic acid molecules is directly proportional, i.e., the lower the pH value, the greater the number of dissociated acids. Therefore, the pH value tends to decrease as the acidity increases.
The results obtained in the present study were similar to those reported by Carbonaro et al. 27 who worked with different peaches cultivated in the region of São Paulo. They found significant differences in the ascorbic acid content between peaches cultivated in organic versus conventional systems. Brunini et al. 28 observed significant differences in vitamin C content in acerolas produced at different growing sites (243.48 to 818.17 mg.100g -1 ). Denardin et al. 3 found 9.35 mg.100g -1 of ascorbic acid in butiá cultivated in Rio Grande do Sul, while in the present study the value obtained was 1.59 mg.100g -1 , demonstrating the influence of region of cultivation on vitamin C content. When evaluating fruits of the Amazon, Canuto et al. 4 obtained lower values than those observed in this study for bacuri (0.20 mg.100g -1 ) and yellow mombim (0.30 mg.100g -1 ), and higher results for assai (10.10 mg.100g -1 ) and cupuaçu (3.30 mg.100g -1 ).
The fruits with the highest concentration of phenolic compounds were genipap (876.85 mg EAG.100g -1 ) and biri-biri (757.82 mg EAG.100g -1 ), both from Itapetinga, in the state of Bahia. The total flavonoid content (Table 3) in fruits ranged from 0.02 to 77.65 mg of quercetin per 100g -1 of dry pulp (mg EQ.100g -1 ). Among the analyzed fruits, java plum from the Rio Grande do Sul state presented the highest flavonoid content, with 77.65 mg EQ.100g -1 , likely due to the large amount of anthocyanins, a subgroup within the flavonoids present in this fruit. The assai fruit, obtained in Belem in the state of Para, presented 6.60 mg EQ.100g -1 , followed by butia from Pelotas in the Rio Grande do Sul state, with 1.56 mg EQ.100g -1 . The samples that presented the lowest content of these compounds were from jatoba fruit (89.52 mg EAG.100g-1) from Campo Grande, Mato Grosso do Sul state, pequi (109.53 mg EAG.100g -1 ) from Goiania, in the Goias state and sapoti (123.70 mg EAG.100g -1 ) obtained from Fortaleza, in the Ceara state.
In the present study, 72.2% of the fruits presented average phenolic compound content; this percentage comprises all samples from Fortaleza (West Indian gherkin, sapoti, yellow mombim and star fruit), from Belem, Para (assai, pupunha and cupuaçu), in addition to a sample from Itapetinga, Bahia (cajarana) and two from Pelotas, Rio Grande do Sul (jambolão and bush banana). The class with a high content of phenolic compounds corresponded to 22.2% of the samples was represented by two fruits obtained in Pelotas (butia and guava), and two from Itapetinga (genipap and biri-biri). Only jatoba presented a low content of phenolic compounds, in the sample from Campo Grande. Silva et al. 30 found levels close to the present study for Brazilian tropical fruits. Faria et al. 20 reported a content of 91.2 mg of catechin per 100g -1 for the java plum from the city of Pelotas, which is very close that reported in the  present study. Paz et al. 31 reported higher levels than the present study for assai pulp from Fortaleza. Flavonoids are present in the plants as color-conferring pigments, which corroborates the results obtained for the jambolão and assai pulps, which present intense purple-colored pulp. Hoffmann et al. 32 analyzed butia from the town of Capão do Leão (Rio Grande do Sul) and reported higher levels than that obtained in the present study, of 682 mg of catechin per 100g -1 . Silva et al. 26 reported average contents close to the present study in pulp (15.06 -102.45 mg 100 g -1 on dry basis) and by-products (27.47 -207.87 mg 100g -1 on dry basis) of Brazilian tropical fruits. According to Morales-Soto et al. 33 , differences in flavonoid content can be attributed to the extraction procedure of the compounds, differences in agronomic factors (agricultural practices, soil composition), as well as climatic and physiological conditions (phase of maturity). Fruits with a low percentage of flavonoids, such as pequi, caja-manga, persimmon, guava, banana, jatoba, genipap, cajarana, biri-biri, West Indian gherkin, star fruit, pupunha and cupuaçu probably present a higher percentage of phenolic acids.
Rocha et al. 34 investigated the carotenoid content in fruit of the Piauí Cerrado, among them the jatoba. Low values of these components were found, 0.0996 μg.g -1 of lycopene and 1.1068 μg.g -1 of β-carotene. Variation in the carotene composition of fruits has been widely reported in the literature, as a result of the effects of several factors such as genetics, soil type, climate conditions and exposure to sun light 14 .
Both zeaxanthin and lutein are xanthophylls resulting from the hydroxylation process of α-carotene and β-carotene, respectively, which have provitamin A activity; these are important micronutrients for health. During fruit maturation, the conversion of lutein and zeaxanthin causes a decrease in the levels of α-carotene and β-carotene 35 , which was observed for yellow mombim, butia, assai and pupunha.
Other health-promoting effects have been attributed to carotenoids in their action as antioxidants, protecting the body against chronic diseases and certain cancers, macular degeneration, cataracts, neurological disorders, gastric antiulcer activity and a strengthening of the immune system 36 .
According to the simple correlation analysis between variables, phenolic compound content had a strong influence on the antioxidant potential determined by ABTS, presenting a correlation coefficient of 0.7403, and no correlation was observed among the other compounds analyzed. Almeida et al. 21 , when evaluating fruits from the Brazilian Northeast, among them sapoti, observed that the polyphenol content and the antioxidant activity by the ABTS method showed a correlation of 0.94, reinforcing the results obtained in the present study. Barreto et al. 37 evaluated 18 tropical fruits and also observed a high correlation between the phenolic compound content and antioxidant activity (r = 0.998). Between the antioxidant activity by FRAP and the bioactive compounds, no significant correlation was observed between any compound. This is probably due to the bioactive composition of the fruits and the method of determining antioxidant activity 38 .
The oils from butia, bacuri and pupunha, in turn, showed higher levels of saturated fatty acids in comparison with unsaturated fatty acids, and the pits of bacuri and pequi presented a prominence for palmitic acid. According to some studies, the consumption of high amounts of palmitic acid in the diet increases the concentration of plasma cholesterol in the blood, while other studies indicate that this acid has antioxidant properties, preventing atherosclerosis in rats 25 .
Palmitic acid is used in the food industry for the production of margarines and cosmetics, in the manufacturing of creams and soaps and candles, for fabric waterproofing, in paraffin, and also in the form of liquid crystal used in the electronics industry. Kim et al. 39 studied fatty acids present in oil seeds for the application of absorption of medicinal products via cutaneous and concluded that palmitic acid was the most effective.
Palm oil (Elaeis guimeensis) is one of the main sources of palmitic acid, reaching a content of about 40%, which was lower than that found in the bacuri pit (50%) and close to that found in pequi, Butia and pupunha pits, on the other hand, had a high percentage of short chain fatty acids, 62 and 79%, respectively. Among these fatty acids, lauric acid was the majority, presenting 24% in butia and 48% in pupunha.

CONCLUSIONS
From the results obtained in this study it was possible to observe that the different regions and fruits studied presented very distinct characteristics. The cupuaçu pit was rich in unsaturated fatty acids, while the genipap and java plum presented high flavonoid content and the butia had a high concentration of carotenoids. Guava was the fruit with the highest antioxidant activity through the ABTS radical, while the yellow mombim presented the best response when the activity was determined by FRAP. The production and valorization of native fruits is of great importance, since it can boost the availability of fresh fruits with high nutritional value, contributing to food security and the strengthening of the economy of small-scale farming communities, due to the best use of the existing natural resources.