Influence of drying on bioactive compounds and antioxidant activity of fruits of guabiju (Myrcianthes pungens)

The objective of this work was to evaluate the influence of temperature on the content of bioactive compounds of fruits of guabiju (Myrcianthes pungens (O. Berg) D. Legrand). The peel, pulp and seed of fresh guabiju were analyzed in relation to physical-chemical composition, metals, color, phenolic compounds, flavonoids, anthocyanins, vitamin C and antioxidant activity. On the dehydrated samples at temperature of 60 °C, where also determined the moisture and water activity. The fractions of the fruit showed high amounts of metals. After drying, moisture of 1.3, 1.0 and 0.9% were observed for peel, pulp and seed and water activity of 0.44 to 0.54. All the samples darkened, with less variation in the dehydrated peel (∆E 9.2). The samples showed high values of bioactive compounds, and in the fresh peel were observed higher levels of phenolic compounds (8459.8 mg EGA/100g dry extract), anthocyanins (152.0 mg/100 dry extract) and vitamin C (222.9 mg/100g) and on the dehydrated seed higher value of flavonoids (7480.7 mg EQ/100g dry extract). There was 86.3% degradation of anthocyanins in the dehydrated peel. The best values of antioxidant activities were obtained for the dehydrated peel (IC50 1.37 mg/mL), seed (IC50 1.49 mg/mL) and in the fresh peel (IC50 1.41 mg/mL).


Introduction
Brazil has great plant biodiversity (Castuera- Oliveira et al., 2020;Moreira-Araújo et al., 2019), including high number of fruit trees (Schiassi et al., 2018;Vergara et al., 2018;Verruck et al., 2018), with a great variety of antioxidant compounds in its fruits and large number of species with potential of use in the food industry (Neri-Numa et al., 2018).
The drying of vegetable foods promotes a reduction of the moisture content, decreasing the water activity, preventing the deterioration caused by microorganisms (Mahayothee et al., 2020), providing greater product diversity, reducing volume and increasing product life (Araujo et al., 2020;Salimi and Hoseinnia, 2020;Landim et al., 2016;Nunes et al., 2020). However, it can compromise the taste and nutritional quality of the food, due to the thermal sensitivity of the bioactive compounds (Kwiatkowski et al., 2016), besides promoting browning reactions depending on the temperature and time used in the drying process (Kwiatkowski et al., 2016;Salimi & Hoseinnia, 2020).
Therefore, as well as the pulp, the peels and seeds of fruit also have a good amount of bioactive compounds, and generally are not consumed or used, generating waste (Paludo et al., 2019;Teles et al., 2018), both can be subjected to the extraction of these compounds and used in foods (Banerjee et al., 2017;Paludo et al., 2019).
Guabiju (Myrcianthes pungens) is still little explored commercially, and there are few studies in the literature, and no work has been found comparing the fractions of the peel, pulp and seed of fruits, before and after drying. In this context, this work aimed to evaluate the influence of drying on the content of bioactive compounds and antioxidant activity of peel, pulp and seed extracts of guabiju (Myrcianthes pungens (O. Berg) D. Legrand).

Harvesting, selection, washing, sanitization and storage of fruits
The fruits of guabiju (Myrcianthes pungens (O. Berg) D. Legrand) were harvested between January and February 2018, in the coordinate 27º 56 '12 "S and 52º 25' 35" W. A sample of branch with leaves and fruits was saved in the Herbário Pr.
After harvest, the fruits were manually selected and washed in drinking water. Then they were immersed in NaClO solution at 200 ppm for 15 min. Afterwards, the fruits were washed again and dried on paper towels, packed in polyethylene packaging and subjected to freezing at -20 ºC. For the analysis, the fruits were separated into peel, pulp and seed. All processes took place in the absence of light.

Characterization of the fresh guabiju fractions
The peel, pulp and seed of fresh guabiju were analyzed in relation to moisture, fixed mineral residue, soluble solids, lipids, protein, carbohydrates, crude fiber, pH, metal content, color parameter, content of compounds phenolic, flavonoids, vitamin C and antioxidant activity. The anthocyanin content was analyzed only in the peel, due to the absence of characteristic pigmentation in the pulp and seed. Research, Society and Development, v. 10, n. 8, e5510817024, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i8.17024

Drying of guabiju fractions
The drying of peel, pulp and seed of guabiju was carried out individually in an oven with air circulation (Marconi, MA 037, Brazil). Approximately 5 g of each sample were used, placed separately and dried at 60 °C until constant weight.
After drying, were determined the water activity, moisture, color, phenolic compounds, flavonoids, vitamin C and antioxidant activity of peel, pulp and seed, and anthocyanin content, determined only from the peel.

Physical-chemical characterization
The analyzes of moisture, fixed mineral residue, soluble solids, lipids, protein, carbohydrates, crude dietary fiber, pH and metals were carried out according to the methodology of the Manual of Analytical Standards of Adolfo Lutz Institute (Zenebon et al., 2008). Water activity of peel, pulp and seed of guabiju were determined using AquaLab (AquaLab model 4TE, USA). The moisture content was performed using the infrared moisture analyzer (Mars / ID-200 -Brazil), at 105 ° C, using approximately 3 g of samples.
To evaluate the color parameters a Minolta colorimeter (CR-400, Osaka, Japan) was used, where the coordinate L* represents sample brightness ranging from 0 (dark) to 100 (light), a* indicates chromaticity tending from green (-80) to red (+100) and b* shows chromaticity ranging from blue (-50) to yellow (+ 70). To calculate the total color difference (ΔE) the Eq.

Determination of bioactive compounds
The antioxidant activity, phenolic compounds and flavonoids, were determined on fresh and dried extracts of peel, pulp and seed of guabiju. The fresh and dehydrated peel and seed were crushed with the aid of a Mixer (Britânia), protected from light. The fresh and dehydrated pulp were macerated in order to increase the contact area.
The extraction of bioactive components was carried out by means of successive extractions using ethyl alcohol (99%).
The sample was left in contact with 50 mL of solvent for 12 h followed by filtration. The filtrate was kept away from light, and the sample retained in the filter was subjected to further extraction 2 more times and the filtrate (150 mL) was passed to a 250 mL round bottom flask, previously covered with aluminum foil. Subsequently, the extracts were rotated in a rotary vacuum evaporator (Marconi MA 120) at 60 ºC. The concentrated product was lyophilized, weighed and stored at 4 °C until use.
The content of total phenolic compounds was carried out by the Folin-Ciocalteau method with modifications (Singleton et al., 1999). In a test tube 0.5 ml of sample of ethanol extracts were added in the concentration of 5 mg/mL, 2.5 ml of the reagent Folin-Ciocalteau (diluted 1:10 v/v) and 2 mL of 4% of sodium carbonate (w/v). The tubes were shaken and stored for 2 h at room temperature protected from light. After, the absorbance was read on a spectrophotometer (Logen Scientific UV/VIS -LS7052) at 760 nm. The content of phenolic compounds was obtained by linear regression analysis, from a standard curve using gallic acid with concentrations of 1 to 100 μg/mL. The results were expressed in milligrams equivalent of gallic acid per 100 grams of extract (mg EGA/100g).
The flavonoid content was obtained according to the method described by Garrido et al. (2013), with adaptations. In a test tube 0.5 mL of sample of ethanol extracts on the concentration of 5 mg/mL, were added 4.3 ml of 70% ethanol (v/v), 0.1 mL of 10% aluminum nitrate (w/v) and 0.1 mL of 10% potassium acetate (w/v). The tubes were shaken and stored at room temperature, protected from light, for 40 min. Then the absorbance was read on a spectrophotometer (Logen Scientific UV/VIS using quercetin with concentrations of 1 to 100 μg/mL. The results were expressed in milligrams equivalent of quercetin per 100 grams of extract (mg EQ/100g).
The extraction of total anthocyanins was carried out following the methodology of Medina et al. (2011) with adaptations. 5 g of sample where packed in Falcon tubes of 25 mL, which were covered with aluminum foil. Then, 15 mL of the solvent at 4 °C (containing 0.1% HCl P.A.) was added. The extraction took place light-free, under refrigeration (at 4 ± 1 °C), for 2 h. After the time, the samples (MPW-351R) were centrifuged at 8000 rpm for 15 min under refrigeration (at 4 ± 1 °C). The supernatant was collected and the precipitate was washed with 5 ml of solvent, after centrifugation using the same parameters as the first operation. Then, the extract was subjected to vacuum filtration in Whatmann paper filter number 01 and Buchner funnel, with the standardization of the volume of each sample to 20 mL. Subsequently, the extracts were packed in flasks covered with aluminum foil and sent for the quantification of anthocyanins through colorimetric analysis.
The anthocyanins were quantified according to the method described by Fetter et al. (2010), where the absorbance reading was performed at 535 nm, in a spectrophotometer (Logen Scientific UV/VIS -LS7052), using the solvent as white.
The anthocyanin content (AT) was obtained according to Eq. (2): Where: ε is the molar absorptivity coefficient of the species; A is the absorbance of the sample; c is the molar concentration of the species; ℓ is the length traversed by the radiation beam (optical path).
The extraction and quantification of anthocyanins of pulp and seed were not determined due to the absence of pigmentation.
To determine the vitamin C content of the samples (IAL, 2008), 5 g of sample were weighed, added 50 mL of distilled water and crushed with the aid of a mixer (Britânia), transferred to Erlenmeyer of 250 mL. After, 10 mL of sulfuric acid solution (20% v/v) was added, then the content was homogenized and filtered into another Erlenmeyer, washing the filter with water and then with 10 mL of sulfuric acid solution (20% v/v). After, 1 mL of potassium iodide solution (10%) and 1 mL of starch solution 1% (m/v) were added. Then was titrate with 0.0002 M of potassium iodate solution until blue color.
The vitamin C content was obtained according to Eq. (3): Where: V = volume of iodate spent on titration; F = 0.08806 for KIO3 0.0002 mol/L; P = g or mL of sample.
The antioxidant activity of ethanol extract was determined by DPPH analysis. This methodology is based on the measurement of the extinction of the absorption of the radical 2,2-diphenyl-1-picryl hydrazil (DPPH) at 515 nm, according to After evaluating the ideal concentration range, the extract concentration needed to capture 50% of the free radical DPPH (IC50) was calculated by linear regression analysis (Silvestri et al., 2010).

Statistical analysis
All experiments were carried out in triplicate and the results of the analyzes were statistically treated by analysis of variance (ANOVA), followed by comparison of the averages by the Tukey´s or student's t test using Statistica software version 5.0, with 95% confidence level. The values obtained in the present study are in agreement with those described by Assumpção et al. (2017) for the moisture of peel (71.66%), pulp (84.06%) and seed (40.81%) of guabiju. Seraglio et al. (2018) describe moisture content between 83.21 to 83.19 g/100g in fresh guabiju fruits. The ash content found in the present study was lower than that described by Assumpção et al. (2017), 3.96, 3.39 and 3.55 for peel, pulp and seed of guabiju, respectively.

Physical-chemical composition and metals of fresh guabiju fractions
The levels of carbohydrates and lipids were higher, and the proteins levels were lower than those described by Assumpção et al. (2017) in guabiju. The guabiju could be compared to other native species, and can be considered a protein fruit, as well as araçá-pitanga, pêssego-do-mato, among others (Kinupp & De Barros, 2008).
The pH found for peel, pulp and seed (5.17, 5.37 and 4.96) were higher than those observed in jabuticaba (3.68 to 3.80) and red araçá (3.31) and similar to those of fresh guabiju fruit (5.19 to 5.32) and freeze-dried guabiju fruit obtained by Seraglio Guabiju has a higher pH which is good for juices, as described by Massaguer et al. (2014). However, due to its high pH, it is not indicated for the formation of gels, since when developing jellies and jams with guabiju, it is necessary to acidify the products.
Thus, guabiju proved to be a considerable source of K in relation to blackberry, red raspberry, strawberry, cherry and blueberry . Potassium intake is associated with lower blood pressure, reduced mortality from stroke and heart disease, therefore, encouraging the consumption of fruits and vegetables source of this mineral can be an important action for the control and prevention of diseases chronic non-communicable diseases (Porto et al., 2014). Table 2 describes the results of water activity, moisture, color parameters Luminosity (L*), Chromaticity (a * and b *) and ∆E of peel, pulp and seed dehydrated at 60ºC. When the brightness parameter (L*) is positive the sample is lighter (seed), and when negative the sample is darker (skin and pulp). As for the chromaticity a*, it varies between the colors green (-) and red (+), and was possible to verify that the peel had a shade closer to green, while the pulp and the seed had a red shade. The chromaticity b* varies between the colors blue (-) and yellow (+), and was observed that all samples analyzed presented a hue close to yellow. The highest value of ∆E obtained was for the seed, followed by the pulp and peel. According to Alves et al. (2008) the higher the ΔE value, the greater the total color difference of the processed product compared to the original product. Nemzer et al. (2018) analyzed the color parameter of blueberry, cherry and strawberry after drying in hot air at 70 °C and observed that samples with a dark hue, such as blueberry, maintained their dark tone during drying, therefore, they presented a lower ∆E (2.89). In addition, the blueberry showed a decrease in L*, thus looking slightly darker. On the other hand, the cherry and the strawberry, presented higher values of ∆E 10.72 and 20.90, respectively.

Water activity, moisture and color parameters of dehydrated guabiju fractions
When analyzing the color of the jabuticaba pulp, it presented L* of 24.23, indicating a dark hue. As for chromaticity, red (+ a *) 15.64 and yellow (+ b *) of 17.25, showing a greater tendency to yellow than to red (Lemos et al., 2019), which corroborates with the result found in this study.
As observed in Table 2, except for the seed, the samples of peel and pulp showed low values of water activity, less than 0.6. As well as, for the moisture analysis that showed low values for the three guabiju fractions after drying.
The drying process reduced the water activity of the seed and pulp, providing stable microbiological conditions, promoting more safety for conservation of the product (Guiné et al., 2018;Mayor and Sereno, 2004).

Evaluation of bioactive compounds
The values for phenolic compounds, flavonoids, anthocyanins and vitamin C of the samples (peel, pulp and seed) fresh and dehydrated at 60 °C are shown in Table 3.  In the present study, there is a significant reduction in the anthocyanins content of dehydrated seed at 60 ºC with 86.3% of degradation compared to fresh peel. Dalla Nora et al. (2014a) also verified degradation of anthocyanins from guabiju (94%) and red araçá (98%) submitted to drying at 70 °C, which was also observed by Fang (2015), which reported that temperatures above 60 ºC, could occur polymerization of the molecule being transformed into chalcone, causing degradation and turning the solution yellow, demonstrating that the thermal processing causes the degradation of anthocyanins.
Because they are sensitive and unstable compounds, during processing, factors such as temperature, heating time, presence of oxygen and light can affect the stability of anthocyanins (Sharif et al., 2020).
The anthocyanin content observed in the present study was higher than that reported by Reis et al. (2016) of 0.8 mg/100g for fresh guabiju (without seed) and 0.29 mg/100 g for guabiju juice, and lower than the values reported by Andrade et al. (2011) for two varieties of guabiju (seedless fruit) ranged from 334 to 531 mg/100 g dry weight.
According to Celant et al. (2016) the total anthocyanin content in blackberry cultivars in 80% ethanol ranged from 6.76 to 9.42 mg of ECG/g fresh weight. However, Casarin et al. (2016) found 89.02 mg ECG//100 g for fresh blackberry and 77.93 mg of ECG//100g for blackberry flour. Goldmeyer et al. (2014) described anthocyanin content in blueberry of 61.67 mg/100 g.
The anthocyanins content of jabuticaba varieties was from 273.67 to 2892.15 mg of ECG//100g of lyophilized sample (Paludo et al., 2019). In conventional extraction acidified with ethanol, Meregalli et al. (2020) found 116.81 mg of ECG//100g in peel of red araçá.
Although not comparable to fruits rich in vitamin C, its values, especially in the peel of guabiju fresh, are high (222.9 mg/100g). According with Mazzoni et al. (2017) the vitamin C content found in the strawberry was 57.98 mg/100g fresh weight.
Two varieties of red orange presented 54.13 mg/100mL and 56.53 mg/100mL of vitamin C (Habibi & Ramezanian, 2017). Lemos et al. (2019) analyzed the vitamin C content of pulp of jabuticaba and acerola and found values of 10.80 and 3704.50 mg/100g, respectively. Table 4 presents the results of IC50, for peel, pulp and seed of guabiju fresh and dehydrated at 60 °C. It is observed that the best IC50 values obtained for antioxidant activity were for fresh peel, as well as for peel and seed dried. Among the biological activities attributed to phenolic compounds, its ability to reduce free radicals stands out due to the antioxidant activity (Jiao et al., 2018;Verruck et al., 2018, Sonawane et al., 2021Amaral et al., 2021). Analyzing the antioxidant activity of dehydrated blueberry, Goldmeyer et al. (2014) showed IC50 values of 3.83 mg/mL. In this way, it was possible to observe that guabiju had an IC50 comparable to blueberries, and can be included in human food because of its antioxidant properties.

Final Considerations
In guabiju samples, the content of K, Ca, Mg, Na, Fe and Al stands out, where the peel shown higher amounts of Ca, Na, Al, Fe, Zn, protein, soluble solids and crude dietary fiber in relation to the pulp and seed. With the drying process, it was observed darkening of all fractions of fruit analyzed, with less variation in the drying of the peel. Drying reduced the levels of total phenolic compounds, flavonoids, anthocyanins and vitamin C in the peel and pulp, however not affected the antioxidant capacity of the extracts. The guabiju extracts, mainly fresh, presented high levels of phenolic compounds, flavonoids, anthocyanins and vitamin C, when compared to other fruits, mainly native. In this sense, it is suggested to future research the extraction of anthocyanins using different solvents and methods, the encapsulation of extracted anthocyanins to improve their conservation, as well as applications in food products.