Extraction of umbu (Spondias tuberosa) seed oil using CO2, ultrasound and conventional methods: Evaluations of composition profiles and antioxidant activities

Accepted Manuscript Title: Extraction of umbu (Spondias tuberosa) seed oil using CO2 , ultrasound and conventional methods: Evaluations of composition profiles and antioxidant activities Authors: Jˆonatas L. Dias, Simone Mazzutti, Julia A.L. de Souza, Sandra R.S. Ferreira, Luiz A.L. Soares, Luiz Stragevitch, Leandro Danielski PII: DOI: Reference:

S0896-8446(18)30613-2 https://doi.org/10.1016/j.supflu.2018.11.011 SUPFLU 4405

To appear in:

J. of Supercritical Fluids

Received date: Revised date: Accepted date:

12 September 2018 12 November 2018 13 November 2018

Please cite this article as: Dias JL, Mazzutti S, de Souza JAL, Ferreira SRS, Soares LAL, Stragevitch L, Danielski L, Extraction of umbu (Spondias tuberosa) seed oil using CO2 , ultrasound and conventional methods: Evaluations of composition profiles and antioxidant activities, The Journal of Supercritical Fluids (2018), https://doi.org/10.1016/j.supflu.2018.11.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Extraction of umbu (Spondias tuberosa) seed oil using CO2, ultrasound and conventional methods: evaluations of composition profiles and antioxidant activities

Jônatas L. Dias a, Simone Mazzutti b, Julia A. L. de Souza c, Sandra R. S.

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Ferreira b, Luiz A. L. Soares c, Luiz Stragevitch a, Leandro Danielski a,*

a

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Department of Chemical Engineering, Federal University of Pernambuco, Av. Prof. Artur de Sá s/n, CEP 50740-521 Recife,PE, Brazil b

Chemical and Food Engineering Department, Federal University of Santa Catarina, Florianópolis, SC, CEP 88040-900, Brazil c

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Department of Pharmaceutical Sciences, Federal University of Pernambuco, Av. Prof. Artur de Sá s/n, CEP 50740-521 Recife,PE, Brazil.

* Corresponding

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Graphical abstract

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Samples

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author. Phone/Fax: +55 81 21267235. E-mail address: [email protected] (L. Danielski).

Spondias tuberosa

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3

Extraction

Bioactive extracts

Supercritical fluid extraction (CO2) Ultrasound-assisted extraction (UAE) Conventional extraction methods

Phenolic content Antioxidant activity Free fatty acids 1

Highlights 

Different methods were applied to recover bioactive compounds from umbu seeds.

Polar extracts showed higher phenolic contents and antioxidant activities.



High correlation found between antioxidant activity and polyphenolic

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

content.

Antioxidant activities found were compared with those of butylated

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

hydroxytoluene.

Integrated approach was an interesting procedure for biomass

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

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valorization.

Abstract

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The fruit of Spondias tuberosa (umbu) is widely consumed in the northeast region of Brazil. In this work, the potential of umbu seeds as source of

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bioactive compounds was evaluated. Different extraction techniques were compared in terms of yield, free-fatty acids composition, total phenolic content

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(TPC) and antioxidant activity (AA). The extraction techniques used were supercritical fluid extraction (SFE) with CO2, performed at 40 °C and pressures from 15 to 30 MPa, and Soxhlet and ultrasound-assisted extraction (UAE) with different solvents. The highest yields were obtained by UAE with ethanol/water

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mixtures and by Soxhlet with ethanol. The higher values for TPC and AA were observed for the extracts obtained with polar solvents. Furthermore, a biorefinery approach considering integrated processes (SFE+UAE) in order to recover polyphenols from the SFE residues has also been investigated and the results showed that combining processes may lead to a most effective valorization of agro-industrial residues. 2

Keywords: Spondias tuberosa; supercritical fluid extraction; ultrasoundassisted extraction; Soxhlet extraction; antioxidants.

1. Introduction

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Spondias tuberosa, popularly known as “umbuzeiro”, is an important tropical plant of the semi-arid northeastern region of Brazil. Its fruits are named

“umbu” and are highly appreciated due to their bittersweet taste [1,2]. These

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fruits are especially used for the production of juices, jellies, food additives, among others [3,4].

Studies about this fruit have reported several biological activities, such as

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antioxidant, antiviral, anti-inflammatory and antimicrobial activities; additionally,

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acetylcholinesterase inhibition and cancer chemopreventive activities were

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reported [5,6]. These previous works have been limited to study the potential use of umbu pulp and there are few studies about the application of the seeds,

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which are agro-industrial residues without defined applicability.

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Fruit agro-industrial residues, such as umbu seeds, are important sources of by-products. Usually, these residues contain high amounts of various bioactive compounds that can be extracted to provide diverse products, like

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nutraceuticals, food additives and pharmaceutical products [7]. Active substances potentially extractable from the targeted residues include sugars,

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polysaccharides, proteins, fibers, natural flavor compounds and several phytochemical compounds, such as free-fatty acids (FFA) and polyphenols [8]. FFA are nonpolar compounds considered as nutraceutical and stimulator

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agents, especially the -6 and -3 fatty acids (linoleic and linolenic acids, respectively). Their application is reported in the literature as important in the prevention of heart diseases, cancer, hypertension and several others [9-11]. Polyphenols have been extensively studied because of their potential as antioxidants [12]. These compounds have one or more aromatic rings with one or more hydroxyl groups. They include a wide variety of molecules and are 3

divided into several classes: phenolic acids, flavonoids, anthocyanins, tannins and stilbenes, according to the number of phenolic rings and structural elements present in the molecules [13]. Experimental studies have indicated that polyphenols are capable of neutralizing free radicals and may play a major role in the prevention of degenerative diseases, such as cancer, diabetes, macular degeneration, and Alzheimer´s and Parkinson´s diseases [14-17].

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Polyphenols and FFA from agro-industrial residues can be obtained by various conventional extraction methods, including Soxhlet, heat reflux and maceration. However, most of these techniques present some drawbacks due

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to the high solvent consumption, the long extraction time required, low extraction yields and possible degradation of target compounds. To overcome these limitations, new extraction procedures have been employed, where the

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most noticeable ones are the supercritical fluid extraction and ultrasound-

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assisted extraction [18].

Supercritical fluid extraction (SFE) offers several operational advantages

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over traditional extraction methods. Supercritical fluids present interesting

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transport properties. They can diffuse easily through solid materials, offering high extraction rates [19]. In particular, SFE using supercritical carbon dioxide

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(SC-CO2) is advantageous because CO2 is nonexplosive, nontoxic and lowcost. Furthermore, since its critical temperature is low, SFE-CO2 avoids the

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degradation of thermolabile substances and the solvent can be completely removed, providing solvent-free extracts [20,21].

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Ultrasound-assisted extraction (UAE) is an emerging technology that has been extensively studied [22-26]. The driving force of UAE is acoustic cavitation, a process that occurs when an ultrasonic wave propagates in a liquid medium. During propagation, series of compressions and rarefactions occur in

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the molecules of the medium and these pressure changes cause the production and collapse of a series of microbubbles. These processes cause rupture and dilution of cell membranes, resulting in improved solvent penetration into the cells and increased mass transfer from the solutes to the solvent [27]. By applying ultrasound, complete extractions can be performed in minutes, with high reproducibility and low amounts of solvent used [28]. 4

Agro-industries generate a considerable amount of residues. In many cases, these residues are disposed in the environment without further exploitation. At the same time, global warming, climate changes and the possible lack of resources in the future have contributed for the development of new productive strategies, focused not only on productivity, but also on process greenness and sustainability. In this context, the biorefinery concept emerged, which can be defined as the development of integrated processes for the

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conversion of biomass into energy and a variety of products, mainly biofuels

and highly added value co-products [29]. The biorefinery concept covers an range

of

combined

technologies

aiming a

full

sustainable

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extensive

transformation of biomass into valuable products. Therefore, the integration of two or more green extraction techniques plays an important role, not only in overcoming the main disadvantages of a single extraction method, but also for

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the sustainable separation of value-added compounds. In this perspective,

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green extraction techniques like SC-CO2 and UAE using green solvents and/or their integration are useful tools to achieve a sustainable utilization of agro-

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industrial biomasses [30].

In this work, umbu seed extracts were studied to: (i) evaluate the

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difference of extraction yields using SFE, UAE and conventional methods; (ii) determine FFA and polyphenol extract contents; (iii) investigate the antioxidant potential of the obtained extracts; (iv) determinate the relationship between the

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antioxidant activity and the content of phenolic compounds; (v) investigate an integrated process for umbu seed biomass valorization based on the biorefinery

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approach.

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2. Materials and methods 2.1. Chemicals All solvents and reagents were of analytical grade. DPPH (1,1-diphenyl2-picrylhydrazyl) radical, gallic acid (≥98.0%), β-carotene, linoleic acid, Tween 20 and Folin–Ciocalteau phenol reagent were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Chloroform, hexane and ethanol were purchased 5

from Neon (Suzano, Brazil). Anhydrous sodium carbonate was purchased from Lafan Química Fina LTDA (Várzea Paulista, Brazil). Dry carbon dioxide (99.9%) was purchased from White Martins (São Paulo, Brazil). 2.2. Raw material S. tuberosa fruits were kindly donated by a local producer (Alagoinha, PE, Brazil, approximate geographical coordinates: 8° 27’ S, 36° 46’ W). The

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seeds were manually separated and dried using an air-circulation oven (Lucadema, Model 82/480, Brazil) at 40 °C during 120 h. Then, the dried seeds

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were grounded in a knife mill (Marconi, Model MA340, Brazil) and their moisture was determined by the oven drying method [31].

Particle diameter and porosity of the fixed bed formed by grounded

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seeds and used in all SFE experiments were determined. The mean particle diameter was obtained by scanning electron microscopy (Shimadzu, Model SS-

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550S, Kyoto, Japan). Additionally, the real density of the solid (ρr) was

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determined by pycnometry with helium displacement (Micromeritics, Model AccuPyc II1340, Norcross, USA). The bed porosity (ε) was calculated

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considering the apparent density (ρa) using Eq. 1:

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𝜀 = 1−

𝜌𝑎 ⁄𝜌𝑟

(1)

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2.3. Supercritical fluid extraction (SFE)

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The SFE-CO2 experiments were performed in the extraction unit outlined in Fig. 1 and described previously in the literature [32]. This unit contains a CO2 reservoir, a thermostatic bath (Microquímica, MQBTZ99–20, SC, Brazil) kept at −5 °C, an air driven piston pump (Maximator M0111, Nordhausen, Germany)

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and a stainless steel jacketed extractor. In addition, a thermostatic bath (Microquímica, MQBTC99-20, SC, Brazil) was used to control the temperature of the extraction cell. Furthermore, the operational pressure and solvent flow rate were controlled by high-pressure valves, manometers and one flowmeter. Approximately 25 g of the raw material was placed inside the extraction column and the empty space was filled with cotton and glass beads. The SFE 6

experiments were performed in dynamic mode at temperature of 40 °C and pressures of 15, 20 and 30 MPa at constant solvent flow rate (11.66 ± 2 g CO2/min). The extraction time was set at 180 min according to one previous kinetic extraction curve carried out at 20 MPa, 40 °C (intermediary conditions of experimental design) and 11.66 ± 2 g CO2/min. Static extraction time was not considered for all SFE experiments. Samples were collected in amber flasks previously weighted on an analytical balance and the yield (X0) was calculated

Extracts were stored in amber flasks at -18 °C prior to analysis.

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2.4. Ultrasound-assisted extraction (UAE)

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as the ratio between the extracted mass and the sample mass in dry basis.

The ultrasound-assisted extraction was conducted in an ultrasonic probe apparatus (Ultronique, Model QR 500, Eco-Sonics, Indaiatuba, Brazil). The

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process was carried out with power of 500 W, frequency of 20 kHz and

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solid/solvent ratio of 1:30 (w/v, g/mL) for 4 min. In each experimental run, 5 g of

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the raw material was placed in one erlenmeyer flask with the chosen solvent (ethanol, hexane or ethanol/water mixture (70:30)). Additionally, in order to

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evaluate the potential of the remaining SFE raffinates obtained after SFE experimental runs, ultrasonic treatment was applied to these samples (denoted

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as UAE-SFERaff) using the ethanol/water mixture as solvent. Then, the obtained extracts were filtered under vacuum and the solvent was removed with a rotary

to analysis.

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vacuum evaporator at 40 °C. The obtained extracts were stored at -18 °C prior

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2.5. Conventional extraction Conventional extraction was carried out using the Soxhlet technique

(SOX) with ethanol and hexane as solvents. The procedure consisted of solvent

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dripping over a determined mass of dry sample, in a Soxhlet apparatus for 4 h at the boiling temperature of the used solvent. A solid/solvent ratio of 1:6 (w/v, g/mL) was used. For each experimental condition, approximately 25 g of the raw material were deposited in individual cellulose cartridges and then inserted into the extractor. Finally, solvent removal and storage of the extracts were performed as described previously (Section 2.4). 7

2.6. Chromatographic procedures The identification of the lipid fraction (FFA) present in the extracts was performed by a gas chromatograph with flame ionization detector (Shimadzu,

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Japan) using a DB-5MS column (30 m length × 250 μm diameter × 0.25 μm film thickness - Agilent Technologies, model 7890, USA). The column was heated at 150 °C for 4 min, with a heating slope of 4 °C /min up to 280 °C, keeping it

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constant for 5 min. FFA were identified by comparing the peak retention times

observed in the samples with standards (FAME Supelco™ mix C4-C24, Bellefonte, PA, USA). FFA were quantified using the peak area normalization

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method and the results were expressed as mass percentage.

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2.7. Total phenolic content (TPC)

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The determination of the total phenolic compounds (TPC) was performed

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using the Folin-Ciocalteau method [33], with some modifications. Initially, the extracts were diluted in ethanol to a final concentration of 10 mg/L. The reaction mixture was composed by 0.79 mL of distilled water, 0.01 mL of extract

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solution, 0.5 mL of Folin-Ciocalteau reagent and 0.15 μL of 20% (w/v, g/mL) sodium carbonate. The tubes containing the reaction medium were shaked,

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incubated at ambient temperature in the absence of light for 2 h and then their absorbances were measured at 765 nm. TPC was measured using a standard

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calibration curve, prepared previously using gallic acid from 0.05 to 0.50 mg/mL as a standard phenolic. TPC was expressed as milligrams of gallic acid equivalent (GAE) per gram of extract. The runs were carried out in triplicate.

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2.8. β-carotene linoleic acid bleaching method The antioxidant potential of the umbu seed extracts was measured using

the β-carotene-linoleic acid bleaching method (BBM) [34]. In brief, linoleic acid (40 mg), tween 20 (400 mg) and β-carotene (3.4 mg) were mixed with chloroform. Chloroform was removed with a rotary vacuum evaporator at 50 °C and distilled water (100 mL) was added. One milliliter of the β-carotene-linoleic 8

acid emulsion was transferred into several flasks containing 0.04 mL of extract (1666,67 µg/mL in ethanol). The flasks were incubated in a water bath at 50 °C for 2 h and the bleaching of the system was monitored at 470 nm. For each sample, a blank was prepared, adding 0.04 mL of extract and 1 mL of blank emulsion (emulsion without β-carotene). In addition, a control sample was prepared by adding 40 μL of ethanol and 1 mL of β-carotene emulsion. The

inhibition, AA, and was calculated according to Eq. (2):

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antioxidant activity was expressed as percentage of β-carotene bleaching

(2)

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𝐴𝐴(%) = [( 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑏𝑙𝑒𝑎𝑐ℎ𝑖𝑛𝑔 − 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑙𝑒𝑎𝑐ℎ𝑖𝑛𝑔 )⁄(𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑏𝑙𝑒𝑎𝑐ℎ𝑖𝑛𝑔)] × 100

2.9. DPPH free radical scavenging assay

The antioxidant capacity of umbu seed extracts was also evaluated by

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the DPPH free radical scavenging method described by Mensor et al. [35], with some modifications. Briefly, ethanolic solutions of the extracts were prepared to

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final concentrations ranging from 5 μg/mL to 30 mg/mL. A volume of 0.71 mL of

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each extract solution was added to 0.29 mL of ethanol solution of DPPH (0.3

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mmol/L). After 30 min of reaction at room temperature in the absence of light, absorbance readings were performed at 518 nm. The percentage antioxidant

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activity, AA, was calculated using Eq. (3): 𝐴𝐴(%) = [( 1 − (𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 − 𝑏𝑙𝑎𝑛𝑘 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 )⁄(𝑏𝑙𝑎𝑛𝑘 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒)] × 100

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(3)

The antioxidant activity was expressed as the amount of antioxidant necessary

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to decrease the initial DPPH concentration in 50% (EC50). 2.10. Statistical analysis The analytical procedures were carried out in triplicate and the results

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presented correspond to the average followed by standard deviation. One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to determine significant differences between treatments (p < 0.05). In addition, Pearson’s correlation test was used to evaluate the relationship between TPC and antioxidant activity. All statistical analyses were performed with the statistical software Minitab 17™ (Minitab Inc., State College, PA, USA). 9

3. Results and discussion 3.1. Yield (X0) of non-conventional and conventional extraction methods For extraction characterization purposes, moisture, particle size and the porosity of the fixed bed of particles for SFE experiments were determined. The

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moisture of the dried substrate was 8.52 ± 0.04%(w/w) and the mean particle size was 8.73 ± 0.04 μm. According to Eq. 1, the bed porosity was calculated as 0.8, considering a real particle density of 1.4073 g/cm3 and an apparent bed

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density of 0.281 g/cm3.

As described in Section 2.3, one previous SFE kinetic assay was performed at 20 MPa, 40 °C (intermediary conditions of experimental design)

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for approximately 7 h with a flowrate of 11.66 ± 2 g CO2/min. From the analysis

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of the kinetic curve, the fast and slow extraction stages were observed. In the constant extraction rate (CER) step, the solute present in the particle surface is

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transferred by convection to the solvent. That was observed by data analysis for

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the first 40 min of extraction. The falling extraction rate (FER) step is characterized by a reduction of solute on the particle surface and occurrence of

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two simultaneous mass transfer mechanisms (convection and diffusion). FER period was found to take place between 40 and 90 min. In the last stage of

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extraction, called the diffusion-controlled step, the curve approaches the maximum content of extractable solute value (X0) and, in this experiment, it was achieved at approximately 180 min. Therefore, based on the kinetic assay, the

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SFE time for global yield extraction was determined as 180 min for all experiments.

The yields obtained by the extraction techniques studied are shown in

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Table 1 and compared in Fig. 2. As can be seen, it was possible to obtain reasonable amounts of extracts for all techniques using polar and nonpolar solvents, indicating that umbu seeds are rich in various compounds with different polarities. [ Please, insert Table 1 here - before reference [38]. Otherwise, references´ citations will be affected due to the use of footnotes in Table 1 ] 10

The UAE-SFERaff extract showed the highest yield (10.9%), almost twice the value obtained by Omena et al. [38] (5.6%) for the extraction by maceration with ethanol. This result may have occurred due to shear forces, turbulence, and erosion typical of UAE processes. These effects enhance the release of extractable compounds of the vegetable matrix. Chemat et al. [39] presented a series of SEM microscopies that evidence these effects on the surface of different vegetal substrates. In addition, the use of a solvent with higher polarity

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may contribute to increase the extraction yields.

The yield obtained by UAE with previous extraction stage with SC-CO2

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(UAE-SFERaff) was significantly higher than the obtained applying only UAE with

the same solvent. This difference could be explained by two main effects: (1) removal of nonpolar compounds such as waxes and resins in the previous step

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may increase the extraction yield of polar compounds [40,41]; (2) high pressure techniques may cause structural modifications in the plant matrix, mainly due to

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the depressurising step what can lead to increase in extraction yields [42].

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These results demonstrate that integration of processes is interesting to obtain

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two different extracts (lipid-rich and phenolic-rich) and changes in the structure of the raw material during the first extraction stage benefit the second step.

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The increase in pressure (in the studied levels) did not significantly affect the yield at constant temperature as the SFE extracts presented similar yields.

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However, it has been reported that the extraction yield can increase with an increase in pressure at constant temperature, due to the increase in solvent power which is closely related to the variation of solvent density [43]. This

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behavior may have occurred because the time chosen was long enough to extract the solutes almost completely, leading to results that are not statistically

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different. Similar results were reported in some works [44-46]. As the SFE extracts presented similar yields, the choice of which

raffinate would be used in a later UAE step was based on the subjective cost that the SFE step would entail to the integrated process. Thus, the raffinate of the milder pressure condition (15 MPa) was used. In comparison with the other results, Soxhlet extraction with ethanol presented the second highest yield (9.1%). The high-temperature conditions, 11

solvent recycling and solvent/solute interactions of the Soxhlet method contributed to the higher solubilization of raw material components. Moreover, as the process was carried out at the boiling temperature of the solvent, the surface tension and viscosity were lower than those at the lower temperature. Therefore, the solvent was able to reach the active sites within the vegetal matrix more easily, increasing oil solubilization [47]. The Soxhlet extraction presents drawbacks when compared to other techniques. It is a time-consuming

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method with the use of a considerable amount of solvent. As the process is carried out at high temperatures, thermal decomposition of thermolabile target

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compounds may occur. Additionally, a further separation procedure must be used to eliminate the solvent from the obtained extracts [48]. 3.2. Free-fatty acids (FFA) chemical profiles

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In order to evaluate the SFE operational conditions at 40 °C and to

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compare the efficiencies of the extraction techniques investigated in this work (UAE, SOX and SFE), umbu seed oil compositions were investigated. The

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obtained extracts were analyzed and the lipidic fraction was determined as a

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function of the extraction methods and operational conditions employed. The FFA profiles were obtained by gas chromatography and are presented in Table

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2. The lipidic fraction analysis of umbu seed extracts showed the presence of palmitic, stearic, oleic, linoleic and linolenic acids, with the exception of extracts

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obtained with the mixture ethanol/water (polar mixture). The oleic and linoleic acids constituted the majority (mass basis) of the identified components. In some cases, there were statistically significant differences in the extract

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compositions obtained. However, these variations were too small when a significance of 5% was considered (Tukey´s test). Similar behavior was reported by Guindani et al. [49] in the investigation of the FFA profiles of chia

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seed pie.

As can be seen in Table 3, umbu seed extracts presented a high

percentage of unsaturated fatty acids (UFA, 70-72%), with polyunsaturated fatty acids (PUFA) as major components (37-39%), all in a mass basis. In addition, it was possible to observe a close relationship between saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and PUFA. The quality and 12

digestibility of edible vegetable oils are determined by the UFA composition, especially by the content of essential fatty acids (linoleic and linolenic acids) [50]. Dubois et al. [51] classified 80 vegetable oils in relation to FFA profile. According to this classification, umbu seed extracts present a profile similar to several vegetable oils used in the food industry, such as corn, sunflower, wheat germ, quinoa and sesame. When comparing the ratio between -6 and -3 FFA (Table 3), umbu seed extracts presented values ranging from 13.4 to 18.0.

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These values were significantly lower than those reported by Orsavova et al. [52] for various vegetable oils (saffron, grape, sunflower, wheat germ, pumpkin

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seed, sesame and rice bran). The ingestion of oils with low ratios between -6

and -3 fatty acids is related to the decrease in the predisposition of diverse pathologies [9-11].

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3.3. Total phenolic content (TPC) and antioxidant activity

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The determination of TPC is important to evaluate the antioxidant activity

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of agro-industrial residues, since these are generally highly referred as sources of antioxidants [53]. The antioxidant activity evaluation of plant extracts using

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only one method is a challenge. A single antioxidant may act in a system through several mechanisms and its effectiveness can be different against

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different radicals [54]. In order to assess the antioxidant potential of the umbu seed extracts, two in vitro antioxidant methods (DPPH free radical and BBM)

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were employed. Besides these methods, TPC was also measured. Table 4 presents the antioxidant activities and TPC results of umbu seed

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extracts obtained by conventional and non-conventional extraction methods. The effects of the extraction methods employed in this work and solvent polarities can be observed in Fig. 2. TPC values ranged from 2.5 to 76.0 mg GAE/g extract. Those values were lower than the ones reported by Omena et

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al. [38] obtained by maceration of umbu seeds (202.0 mg GAE/g extract). However, the TPC content can vary significantly in the same plant species as a response to the different abiotic factors. According to Akula and Ravishankar [55], the synthesis and accumulation of secondary metabolites in plants are affected by several abiotic factors, such as temperature, moisture, salinity and soil alkalinity. Therefore, the simple comparison of quantitative results should be 13

done with caution. In addition, umbu seeds were shown to be richer in phenolic compounds than its pulp. Rufino et al. [56] found a TPC value of 0.90 mg GAE/g extract, slightly lower than the values observed in this work. Low TPC values were observed in all extracts obtained with hexane, indicating that a significant part of the phenolic compounds present in the umbu seed are polar compounds. These results are similar to those found by Razali et

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al. [57], where the effect of several solvents on TPC values of Tamarindus indica L was evaluated. For all plant fractions studied, TPC values were lower

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when hexane was used as solvent.

The supercritical extracts presented low TPC, because the solubility of phenolic compounds in SC-CO2 is usually low. Under the conditions studied, the solubility of several phenols found in plant matrices (protocatechinic acid, p-

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coumaric acid, caffeic acid, ferulic acid, methyl gallate and (-)-epicatechin) is in

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the order of 10-7 mole fraction [58-61].

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With respect to the processes carried out by ultrasound using the solvent

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ethanol/water mixture, a slight decrease was observed in the TPC of raffinate from SFE at 15 MPa (UAE-SFERaff: 73.0 mg GAE/g extract) relative to the sample without the SFE previous step (76.2 mg GAE/g extract). This finding can

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possibly be explained by the observed standard deviations, although the removal of some nonpolar polyphenols from the plant material at the SFE stage

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could also have contributed to the difference. The AA from the βcarotene/linoleic acid method (Table 4) showed that the antioxidant potential

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increased with increasing the polarity of the solvent for the same extraction process. This trend suggested that AA is somehow dependent on the presence of polar substances in the extracts. Oliveira et al. [62] found similar behavior in

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their study with passion fruit seeds and seed cake. In general, the extracts obtained by SFE presented low antioxidant

activity. Carbon dioxide, due to its nonpolar characteristic, does not favor the solubilization of phenolic compounds of intermediate to high polarity. As a result, extracts obtained by SFE-CO2 were poor in these compounds, which may explain the low antioxidant potential of these extracts [63]. 14

Regarding to the antioxidant activity determined by the DPPH method, extracts obtained by ultrasonic extraction showed the lowest values of EC 50, thus, presented the highest antioxidant activities. These values were lower than 173.37 µg/mL reported by Omena et al. [38] and obtained for umbu seed extracts via maceration. Furthermore, it was observed that the extracts obtained with polar solvents showed the lowest EC50 values, i.e., higher antioxidant activities. This was another evidence that the antioxidant activity of extracts is

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strongly dependent on the presence of polar compounds. This trend was also reported by Andrade et al. [64] in the evaluation of the antioxidant potential of

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spent and husk coffee extracts obtained by SFE, UAE and Soxhlet. In all methods evaluated, increased solvent polarities were accompanied by increased TPC values and increased antioxidant activities.

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The EC50 values of extracts obtained by UAE and Soxhlet with polar solvents were lower than the results obtained for the commercial antioxidant

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butylated hydroxytoluene (BHT). The measured EC50 value of this standard

A

antioxidant was 305 ± 0.3 μg/mL. However, the antioxidant performance of

M

these extracts was not superior to BHT when using the β-carotene/linoleic acid (BBM) method; i.e., the measured AA of BHT was 100%, while the highest AA

ED

reported for umbu seed extracts was around 71%. Apparently, these extracts present a higher antioxidant activity against hydrophilic radicals (like DPPH) than lipophilic radicals, such as those generated during oxidation of linoleic acid

PT

(BBM method). According to Tan and Lim [65], a single antioxidant may, in some cases, act by multiple mechanisms in a single system or by a different

CC E

single mechanism depending on the reaction system. Therefore, their efficacy against different radical or oxidant sources can be different.

A

3.4. Correlation between TPC and antioxidant activity As presented in Section 3.3, the antioxidant activity of the extracts is

somehow dependent on the presence of polar substances in the extracts. In fact, some studies have shown a clear correlation between antioxidant activity and TPC, which are mostly polar [66-68]. The interrelation between antioxidant activities and TPC has been performed using Pearson’s correlation test (Table 5). According to Table 5, a 15

high Pearson´s correlation coefficient (0.967) was found between the antioxidant activity values by the β-carotene/linoleic acid method and the TPC. The magnitude of this value indicates a strong correlation between these variables. These results are consistent with the results reported by Alu’datt et al. [69] who studied the correlation between antioxidant activity and phenolic content in thyme.

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The correlation between TPC and EC50 showed a low negative intensity value (-0.728), indicating a decrease in EC50 value with an increase in the

amount of TPC in the extracts. As the relationship between EC50 and

SC R

antioxidant activity is inversely proportional, the correlation indicates increase in antioxidant activity with increase in polyphenols content. Dutra et al. [70] reported similar behavior for propolis extracts.

U

Additionally, in order to verify a quantitative relation between AA(%) and

N

TPC, a linear approach has been performed considering BBM and DPPH

A

methods, and the resulting equations are, respectively (Eqs. 4 and 5):

M

𝐴𝐴(%) = 0.729 ∙ 𝑇𝑃𝐶 + 17.57

ED

𝐸𝐶50 = −179.64 ∙ 𝑇𝑃𝐶 + 11711.59 with TPC expressed in mg GAE/g extract, EC50 in g/mL, and

(4) (5) AA(%) in

PT

percentage. R2 values obtained were 0.9267 and 0.5298 for Eqs. 4 and 5, respectively.

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In the literature, some authors have found weak correlations between

TPC and antioxidant activity [71-73]. The weak correlation can be attributed to the presence of some non-phenolic compounds which can react with the Folin-

A

Ciocalteu reagent but which are not effective as free radical scavengers (citric acid, ferrous sulfate, d-glucose), interfering in the correlation between TPC and antioxidant activity of some extracts [74]. This may be one explanation for the low R2 value obtained by Eq. 5. Differences in phenolic profiles (type of phenolics present and their relative proportions), synergism/antagonism among antioxidants, make the 16

antioxidant activity of some extracts not dependent only on phenolic concentration, but also on the structure and interaction between antioxidants [75,76]. However, the significant correlations obtained in this work support the hypothesis that the phenolic compounds present in the umbu seed extracts contribute significantly to the antioxidant potential of the extracts obtained. 3.5. Biorefinery approach

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As reported in previous Sections, after SFE extraction, some valuable

polar compounds such as polyphenols remained in the raffinate (SFE-residue).

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In order to recover them from the residue, a preliminary study was carried out to

evaluate potential integrating technologies wich allow better valorization of the biomass, thus following the concept of biorefinery. The SFE raffinate was selected because SFE could cause minor undesired changes in the raw

U

material compared to other methods, such as Soxhlet. Since it works with mild

N

temperature conditions, reactions like oxidation and degradation would be avoided and the extracts may retain the natural characteristics of the biomass,

M

and food applications.

A

what could increase their potential for pharmaceutical, cosmetic, nutraceutical

Fig. 3 shows a comparison of the individual steps and SFE+UAE

ED

integrated processes in terms of oil yield and TPC. The Fig. 3 corroborated the results and interpretations taken in the previous Sections (global extraction

PT

yield, total phenolic content and antioxidant activity). The integrated process showed a clear advantage over separated processes. Two different fractions

CC E

were obtained. The oil-rich fraction (6.5%) with similar FFA composition to several valuable food oils and other phenolic-rich (73 mg GAE/g extract) that can be used in multiple applications. Furthermore, the results indicated that the integrated processes can be used to increase the extraction efficiency of the

A

UAE technique, as previously showed. This approach of biorefinery presents an interesting feature. The

combination of SFE and UAE technologies using green solvents, such as SCCO2 and ethanol/water, represents an enhancement compared to the biomass treatment only with SFE. These isolated extraction techniques usually neglect the remaining valuable compounds in the residues. Future studies on the 17

current topic should be undertaken to establish process conditions, which could lead to a most effective utilization of the studied biomass.

4. Conclusions Seed extracts from S. tuberosa were found to have high phenolic content

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and good antioxidant activity. The evaluation of the lipidic fraction evidenced the presence of typical FFA, including its subdivision in saturated and unsaturated

acids (MUFA and PUFA). TPC and AA presented a strong dependence on the

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extraction method and the solvent polarity. The ultrasound-assisted extraction

methods were more suitable to obtain extracts with higher phenolic content and antioxidant activity. A significant correlation was found between phenolic

U

content and antioxidant activity of the extracts. This supports the idea that the antioxidant property of extracts is determined mainly by its phenolic content.

N

Applying SFE and UAE as a combined process is a promising and useful tool to

A

recover selectively hydrophilic (phenolic-rich fraction) and lipophilic compounds

M

(oil-rich fraction) from umbu seeds. Finally, it is important to highlight that some extracts presented higher antioxidant activities than BHT. Hence, these extracts

PT

Acknowledgments

ED

have a potential for substitution of synthetic antioxidants.

The authors wish to acknowledge CNPq and FACEPE for the financial support.

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CETENE is gratefully acknowledged for the realization of FFA chromatographic

A

analysis.

18

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IP T SC R U N A

A

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ED

M

Fig. 1. Schematic diagram of the SFE-CO2 experimental unit.

27

IP T SC R U N A M ED

Fig. 2. Effect of extraction method and solvent polarity on yield and antioxidant

A

CC E

shown).

PT

properties of umbu seed extracts (for SFE-CO2, only the values at 15 MPa are

28

IP T SC R U

N

Fig. 3. Comparison between individual steps and integrated approach. SFE: 15

A

CC E

PT

ED

M

A

MPa/40 °C and UAE: ethanol/water.

29

Table 1. Yields (X0) of S. tuberosa seed extracts.

Solvent

Polarity index A

X0 (%)C

Sox

Hexane

0.1

8.4bc ± 0.50

Ethanol

4.3

9.1b ± 0.90

Hexane

0.1

7.8bcd ± 0.10

Ethanol

4.3

Ethanol/water

6.1

UAE-SFERaff

Ethanol/water

6.1

SFE

Solvent

15 MPa/40 °C

CO2

20 MPa/40 °C

CO2

SC R U N

A

10.9a X0 (%)C 6.5de ± 0.40

ED

M

ρCO2 (kg/m3)B

7.1cd ± 0.10

785

6.4de ± 0.04

CO2

871

7.1cd ± 0.70

CC E

30 MPa/40 °C

5.0e ± 0.40

701

PT

UAE

IP T

Method

Solvent polarity index [36]; B CO2 density from Angus et al. [37]; C Values sharing the same superscripts are not significantly different from each other at a confidence level of 95%.

A

A

30

I N U SC R

Table 2. Lipidic fraction profiles of umbu seed extracts obtained using different extraction methods and conditions.

Palmitic acid

M

A

Exp. Run(1)

FFA content(2) (%, mass basis)

Stearic acid

Oleic acid

Linoleic acid

Linolenic acid

(C18:0)

(C18:1)

(C18:2)

(C18:3)

9.6c

32.4d

35.5c

2.6a ± 0.1

19.9bc

9.5c

32.47d

35.8c

2.5ab

SFE30

19.5d ± 0.2

9.8b ± 0.1

32.7bc ± 0.1

35.6c ± 0.2

2.4ab ± 0.3

USHex

19.6cd ± 0.1

9.9b

32.8b ± 0.1

35.5c ± 0.1

2.1ab ± 0.2

USEtOH

18.5e

8.5d ± 0.1

33.2a ± 0.1

37.0a ± 0.1

2.7a ± 0.3

US70/30

n.d.

n.d.

n.d.

n.d.

n.d.

ED

(C16:0)

19.9c± 0.1

A

CC E

SFE20

PT

SFE15

31

I N U SC R

19.9bc ± 0.1

9.9b ± 0.1

32.4d ± 0.2

35.5c ± 0.1

2.3ab ± 0.3

RAF-US70/30

n.d.

n.d.

n.d.

n.d.

n.d.

A

SOXHex

(1)

A

CC E

PT

ED

M

SFE15: SFE at 15 MPa (40 °C); SFE20: SFE at 20 MPa (40 °C); SFE30: SFE at 30 MPa (40 °C); USHex: UAE with hexane; USEtOH: UAE with ethanol; US70/30: UAE with ethanol/H2O (70:30); RAF-US70/30: SFE15 raffinate submmited to UAE with ethanol/H2O (70:30); SOXHex: SOX with hexane; SOXEtOH: SOX with ethanol. (2) Mean values followed by the same superscripts do not differ from each other using Tukey´s test (5% of significance). n.d.: not detected.

32

I N U SC R

Table 3. FFA contents in relation to the mass percentage of SFA and UFA (MUFA and PUFA), and the ratio ω-6:ω-3.

FFA contents(2) (%)

A

Exp. Run(1)

M

SFA

UFA

MUFA

PUFA

Ratio ω-6:ω-3

70.5b

32.3d

38.2bc ± 0.1

13.4

29.5c

SFE20

29.4c

70.6b

32.4d

38.2bc ± 0.1

14.3 ± 0.5

29.3c ± 0.2

70.7b ± 0.2

32.7bc ± 0.1

38.0cd ± 0.2

14.9 ± 2.2

29.5bc ± 0.1

70.5bc ± 0.1

32.8b ± 0.1

37.7de ± 0.1

16.7 ± 1.8

USEtOH

27.0d ± 0.1

73.0a ± 0.1

33.3a ± 0.1

39.7a ± 0.2

13.7 ± 1.5

US70/30

n.d.

n.d.

n.d.

n.d.

n.d.

SOXHex

29.8ab ± 0.2

70.2cd ± 0.2

32.4d ± 0.2

37.8de ± 0.2

15.8 ± 2.3

SOXEtOH

30.0a

70.0d

32.5cd ± 0.1

37.5e

18.0 ± 0.3

RAF-US70/30

n.d.

n.d.

n.d.

n.d.

n.d.

ED

SFE15

A

CC E

USHex

PT

SFE30

(1)

SFE15: SFE at 15 MPa (40 °C); SFE20: SFE at 20 MPa (40 °C); SFE30: SFE at 30 MPa (40 °C); USHex: UAE with hexane; USEtOH: UAE with ethanol; US70/30: UAE with ethanol/H2O (70:30); RAF-US70/30: SFE15 raffinate submmited to UAE with ethanol/H2O (70:30); SOXHex: SOX with hexane; SOXEtOH: 33

I N U SC R

(2)

Mean values followed by the same superscripts do not differ from each other using Tukey´s test (5% of significance). n.d.: not

A

CC E

PT

ED

M

A

SOX with ethanol. detected.

34

I N U SC R

Table 4. Antioxidant activity for umbu seed extracts.

TPC (mg GAE/g extract)*

AA (%)*

EC50 (µg/mL)*

Hexane

3.3d ± 0.6

20.1de ± 2.2

>5000

Ethanol

33.5b ± 0.9

48.9b ± 1.0

185.1a ± 0.2

Hexane

2.1d ± 0.2

22.9d ± 0.9

>5000

Ethanol

10.0c ± 0.6

32.5c ± 0.4

39.5c ± 0.6

Ethanol/water

76.0a ± 0.7

71.4a ± 3.3

79.9b ± 0.2

Ethanol/water

73.0a ± 4.0

70.5a ± 3.8

180.6a ± 4.8

SFE 15 MPa/40 °C

CO2

2.7d ± 0.6

17.1def ± 1.7

>5000

SFE 20 MPa/40 °C

CO2

2.5d ± 0.1

14.2f ± 0.4

>5000

Solvent

Sox

ED

M

A

Method

CC E

PT

UAE

A

UAE-SFERaff

35

I N U SC R

SFE 30 MPa/40 °C

3.5d ± 0.2

CO2

16.1ef ± 1.8

>5000

A

CC E

PT

ED

M

A

*Values sharing the same superscripts are not significantly different from each other at a confidence level of 95%.

36

AA

EC50

1

0.967

-0.728

AA

0.967

1

-0.770

EC50

-0.728

-0.770

1

A

CC E

PT

ED

M

A

N

U

TPC

SC R

TPC

IP T

Table 5. Pearson’s correlation coefficients among antioxidant capacity parameters and total phenolic content.

37