Evaluation of bioactive compounds from Sapodilla (Manilkara zapota) peel and seeds obtained by ultrasound-assisted technique Evaluación de compuestos bioactivos de cáscara de zapote (Manilkara zapota) y semillas

Sapodilla is an exotic fruit consumed in several countries, which generates a significant amount of waste which can be used as a source of bioactive compounds. In this context, this work aimed to extract bioactive compounds from sapodilla peel and seeds through an ultrasound-assisted technique. This work is an explanatory quantitative research based on laboratory experiments. Extraction was carried out with distilled water, methanol and ethanol at concentrations between 40% and 80%, subjected to ultrasound for 1 h. The highest levels of phenolic compounds were obtained in 40% methanol peel extract (126.0 mg GAE/100 g of residue) and in 80% methanol seed extract (65.3 mg GAE/100 g of residue). In relation to total flavonoids, the highest levels were found in 80% ethanol peel extract (90.0 mg QCE/100 g) and in 80% ethanol seed extract (33.3 mg QCE/100g). The highest antioxidant activity for these extracts was obtained by the ABTS method, around 700.0 μM Trolox/g of residue. Seven polyphenolic compounds were identified and quantified by HPLC, with gallic acid being the major compound, followed by epigallocatechin and catechin. The ultrasound technique was efficient for obtaining bioactive extracts of sapodilla residues with potential for future application as a natural source of bioactive compounds.


Introdution
Sapodilla (Manilkara zapota/Achras zapota) or sapota is the most well-known fruit species in the Sapotaceae family (Junior et al., 2014); native to Central America, it originated in southern Mexico or Central America (Oliveira et al., 2011). In Brazil, Pernambuco is the largest national producer, followed by states such as Bahia, Ceará, Pará, Paraíba, Rio Grande do Norte and Sergipe, mainly in the centre-south region, in municipalities such as Lagarto and Boquim (Junior et al., 2014). In general, sapodilla is consumed in its fresh form, juices, ice cream and jams, while in international industry it is used for the manufacture of sweets, soft drinks, preserves, jams and syrups (Costa et al., 2017).
Fruit processing commonly causes the generation of tons of waste, often not used. As a consequence, over the years, several studies have evaluated the composition of agroindustrial waste in the search for bioactive compounds of industrial interest. Over the years, researchers have determined bioactive compounds such as total phenolics, total flavonoids, anthocyanins and ascorbic acid in sapodilla peel, pulp leftovers and seeds (Silva et al., 2014;Sancho et al., 2015;Singh et al., 2016;Can-Cauich et al., 2017) through the conventional method of extraction with organic solvents and orbital shaking. However, this method is not efficient for the extraction of phenolic compounds present in the residue matrix in bound form. In addition, traditional extraction methods possess drawbacks such as long extraction Research, Society andDevelopment, v. 9, n. 8, e354985158, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5158 4 periods, the necessity of using solvents with high purity, low extraction selectivity, solvent consumption in huge quantities and degradation of heat-labile components (Koçak and Pazır, 2018).
As an alternative, novel techniques such as ultrasound-assisted extraction have been employed. Ultrasound waves are certain types of electromagnetic radiation which propagate through a medium with a frequency range between 20 and 100 MHz by generating compression and expansion (Chemat et al., 2011). Ultrasound extraction is a very simple procedure, where plant material or plant cells are disrupted by the application of ultrasound waves that promote the release of bioactive compounds into the surrounding solvent. The ultrasound extraction technique has advantages such as: shorter operating time, easier handling, reduced temperature, less solvent use and energy savings; and has the potential to increase extraction yields due to cavitation and improved mass transfer phenomena (Sharayeia et al., 2019). In this context, the aim of this work was to extract bioactive compounds from sapodilla peel and seeds using organic solvents associated with an ultrasound-assisted technique.

Metodology
This article is a quantitative explanatory research (Pereira et al., 2018) developed by the first author in the master's thesis under supervision of the third author. In this study, extracts of sapodilla seeds and peel were obtained using the assisted ultrasound technique associated with extraction with organic solvents. Next, the experimental methodologies used for the development of the work will be presented.

Materials
Mature sapodilla fruits were supplied from a commercial market in Aracaju city (Sergipe, Brazil). Ethanol and methanol were acquired from Neon Comercial (São Paulo, Brazil). Aluminium chloride, sodium carbonate, acetic acid, ferric chloride, glucose monohydrate and potassium persulphate were acquired from Dinâmica (Indaiatuba, Brazil).

Treatment of fruits
The fruits were immersed for 10 min in a chlorinated solution at 200 ppm and washed with water. Subsequently, the peel and seeds were separated manually from pulp. The peel and seeds were dried at 50 °C in a drying oven (Pardal, Brazil) for 24 h and crushed in a blender (Safdar et al., 2017 with modifications). The flours of sapodilla peel and seeds were named FPS and FSS, respectively.

Acquisition of extracts
The peel and seed extracts were obtained using 2 g of solid material, 10 mL of distilled water and hydroalcoholic solutions of ethanol at concentrations of 40%, 50%, 60%, 70% and 80% (Valvi et al., 2011). The solutions were placed in a USC-1400A ultrasonic bath (Unique, São Paulo, Brazil) for 60 min at a temperature of 30 °C, 50 kHz frequency and 250 VA of force (Rezende et al., 2017 with modifications). After that, the samples were filtered on filter paper and the supernatants (extracts) were analysed for total phenolic and total flavonoid content.

Determination of total phenolics in the extracts
The total phenolic content (TPC) was determined by a modified Folin-Ciocalteu method using gallic acid as standard (Shetty et al., 1995). In test tubes, 1 mL of extracts was added to 1 mL of 95% ethanol solution, 5 mL of distilled water and 0.5 mL of 1 N Folin-Ciocalteu reagent, followed by homogenisation. Then, 1 mL of 5% (w/v) sodium carbonate solution was added and homogenised, and kept in the dark for 60 min. The absorbances of the samples were determined at 725 nm using each solvent as a blank sample. A calibration curve was constructed from different concentrations of gallic acid (0-150 mg/L) and results Research, Society and Development, v. 9, n. 8, e354985158, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5158 6 expressed in milligrams of gallic acid equivalent (GAE) per 100 g of FPS or FSS on a dry basis (d.b.).

Determination of total flavonoids in the extracts
To determine the total flavonoid content (TFC), extracts (2 mL) were added to 2%

In vitro antioxidant activity of the extracts
The extracts with higher TPC and TFC were analysed for antioxidant activity (AA) by ABTS, DPPH and FRAP methods. The ABTS method was performed according to Nenadis et al. (2004) with modifications, using 30 μL of extract and 3.0 mL of ABTS reagent. The solution was vortexed for 6 min and the absorbance was measured at 734 nm. The calibration curve was obtained with different concentrations of Trolox (100-1600 μmol Trolox/L) and the results expressed as μmol Trolox/g of flour (wet basis). For the DPPH method (Kwon et al., 2006 with modifications), 250 µL of extract was mixed with 1.25 mL of DPPH reagent for 5 min and the absorbance was measured at 517 nm. The calibration curve was constructed using 50-250 μmol/L of Trolox. The blank was 95% ethanol. The results were expressed as μmol Trolox/g of flour (wet basis). For the FRAP method (Thaipong et al., 2006), 90 μL of the extract, 270 μL of distilled water and 2.7 mL of FRAP reagent were mixed, vortexed and maintained at 37 °C in a water bath. After 30 min, the absorbance was measured at 595 nm.

Statistical analysis
The results were expressed as mean ± standard deviation and compared by Tukey's test at a 5% level of significance (p < 0.05), using Sisvar 5.6 software.

Total phenolic and total flavonoid content in the extracts
The TPC was determined for the extracts obtained in different solvents from FPS and FSS ( Figure 1A and 1B). Research, Society and Development, v. 9, n. 8, e354985158, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5158 The solvents used in this study have different polarities; as molecular weight of solvent increase the polarity decrease, the lower the polarity. The order of magnitude in relation to polarity index (PI) is (Snyder, 1974): distilled water (PI = 9.0) > methanol (PI = 6.6) > ethanol (PI = 5.2). Based on the results obtained, the phenolic compounds extracted from sapodilla peel and seeds were of high polarity with affinity for methanol, the compounds Research, Society and Development, v. 9, n. 8, e354985158, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5158 9 from seeds being more polar since they were extracted with 80% methanol. In general, methanol, being highly polar, increases the solubility of bioactive compounds, facilitating extraction (Horincar et al., 2019). Horincar et al. (2019) and Safdar et al. (2017) also obtained greater extraction of bioactive compounds in aubergine and citrus peel with methanol as solvent. Despite of this, the phenolic content in 60% ethanol extract not differed statistically from obtained by 40% methanol extract in PSF. In this case, the use of ethanol would be more interesting, since that this solvent is low cost and the most preferred among alcohols due to its low boiling point, quick recovery and 'generally regarded as safe' status (EFSA, 2011).  (Figures 2A and   2B). Research, Society and Development, v. 9, n. 8, e354985158, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5158 mg QCE/100 g of dry weight residue in sapodilla peel after two extractions with methanol in a shaking incubator (160 rpm) at 25 °C for 24 h. These differences in TFC may be due to the origin, planting and chemical composition of the fruit as well as the extraction method used.

Antioxidant activity of sapodilla residue extracts
The extracts with the highest TPC and TFC (sapodilla peel in 40% methanol and 80% ethanol and seeds in 80% methanol) were analysed for in vitro AA using the ABTS, FRAP and DPPH methods (Table 1). Table 1. Antioxidant activity of selected extracts of sapodilla peel and seeds flours.
Results expressed as mean ± standard deviation (n = 3). a-b Different lowercase letters in the same column indicate significant differences (p <0.05) between the mean values in the same column. Source : this work In this Table, it is important to note which extract showed the highest antioxidant activity. Also compare the values obtained in each method (same column) noting that the same letters mean that the values not differed statistically among them and different letters mean that they differed.
The highest AA values were obtained by the ABTS method; those for 80% methanol seed (702.3 μM of Trolox/g of residue) and 80% ethanol peel (700.7 μM of Trolox/g of residue) extracts did not differ statistically (p < 0.05). This result may be due these extracts having hydrophilic and lipophilic compounds, which are more reactive with the ABTS radical than the other radicals (Gulçin, 2012). The 80% ethanol and 40% methanol peel extracts showed higher AA by FRAP and DPPH methods, respectively, differing from the values obtained for other extracts (p > 0.05).
The AA of the extracts was higher than the values obtained by Almeida et al. (2011) for 60% methanol sapodilla pulp extract (0.99 and 0.17 μM of Trolox/g of fresh pulp, for ABTS and DPPH, respectively) and by Can-Cauich et al. (2017)

Identification and quantification of bioactive compounds in sapodilla residue extracts
Seven polyphenolic compounds were identified in the extracts, three phenolic acids from the subclass of hydroxybenzoic acids (gallic acid, protocatechuic acid and vanillic acid) and four from the flavonoid class (catechin, epigallocatechin, ethyl gallate and epigallocatechin gallate) ( Table 2). In all extracts, gallic acid was the major compound, with the highest concentration (0.065 mg/g dry) in 80% methanol seed extract. Gallic acid (3,4,5-trihydroxybenzoic acid) is considered one of the main phenolic acids, and has attracted the interest of researchers for its antioxidant capacity, and actions such as antimicrobial, antifungal, anticancer and antiinflammatory, among others (Fernandes & Salgado, 2016). Epigallocatechin and catechin were found in the extracts at concentrations of between 0.034 and 0.040 mg/g dry matter and 0.015 and 0.017 mg/g dry matter, respectively. The catechins possesses antimicrobial, antioxidant, neuroprotective, cardioprotective, antitumour and anti-inflammatory effects and vasodilator activity and offer macular protection (Velayutham et al., 2008;Pedro et al., 2019).

Final Considerations and Suggestions
In this work, the bioactive compounds of sapodilla peel and seeds were obtained using different solvents and an ultrasound-assisted technique. Sapodilla peel showed higher TPC and TFC than seeds. Methanol was the more effective solvent for extracting polyphenolic compounds. The extracts with higher TPC and TFC showed higher AA by the ABTS method.
Gallic acid was the major compound found in the extracts, followed by epigallocatechin and catechin. The compounds were efficiently extracted from sapodilla residues by the ultrasound-assisted technique, showing the potential of these extracts to be explored in future studies as sources of bioactive compounds of industrial interest.
In future works, antimicrobial activity and in vivo antioxidant activity of these extracts can be evaluated. In addition, studies on the application of extracts as a source of pharmacological compounds, as well as source of natural additives for food preservation, are suggested.