Açaí (Euterpe oleracea Mart.) Seed Extracts from Different Varieties: A Source of Proanthocyanidins and Eco-Friendly Corrosion Inhibition Activity

Euterpe oleracea Mart. (Arecaceae) is an endogenous palm tree from the Amazon region. Its seeds correspond to 85% of the fruit’s weight, a primary solid residue generated from pulp production, the accumulation of which represents a potential source of pollution and environmental problems. As such, this work aimed to quantify and determine the phytochemical composition of E. oleracea Mart. seeds from purple, white, and BRS-Pará açaí varieties using established analytical methods and also to evaluate it as an eco-friendly corrosion inhibitor. The proanthocyanidin quantification (n-butanol/hydrochloric acid assay) between varieties was 6.4–22.4 (w/w)/dry matter. Extract characterization showed that all varieties are composed of B-type procyanidin with a high mean degree of polymerization (mDP ≥ 10) by different analytical methodologies to ensure the results. The purple açaí extract, which presented 22.4% (w/w) proanthocyanidins/dry matter, was tested against corrosion of carbon steel AISI 1020 in neutral pH. The crude extract (1.0 g/L) was effective in controlling corrosion on the metal surface for 24 h. Our results demonstrated that the extracts rich in polymeric procyanidins obtained from industrial açaí waste could be used to inhibit carbon steel AISI 1020 in neutral pH as an abundant, inexpensive, and green source of corrosion inhibitor.


Introduction
Euterpe oleracea Mart., an endogenous palm tree of the Amazonian biome that grows in flooded areas, produces a dark purple fruit called açaí [1]. Its edible pulp is rich in antioxidants and nutrients to the most impoverished local populations [2] and now commercialized as a functional food [3]. Açaí has high nutritional value due to the predominance of fatty acids and amino acids. However, the antioxidant capacities of high contents of

Results and Discussion
The potential health benefits attributed to açaí seed extracts [29][30][31] highlight how critical comprehensive chemical characterization is. Research on the seed extract's new biological effects is associated with its composition, thus adding value to this residue. Therefore, we started to investigate the chemical composition of varieties of açaí seeds: purple açaí (PA), white açaí (WA), and BRS-Pará (BRS).
The results from the extraction and liquid-liquid partitioning yields are shown in Table 1. The extraction generated 6.99% (w/w), 7.61% (w/w), and 8.04% (w/w) yields for BRS, WA, and PA, respectively. Liquid-liquid partitioning with ethyl acetate and water 1:1 (v/v) can separate PACs by size since only small weighted ones and other polyphenols are soluble in ethyl acetate. The aqueous fractions yielded above 75%, indicating that all varieties exhibited a high number of polymeric PACs. After extraction, the content of PACs was quantified by n-butanol/hydrochloric acid assay following a published protocol [32]. PA exhibited 22.4% (w/w) proanthocyanidins/dry matter, while WA displayed 6.4% (w/w) and BRS 11.5% (w/w). Thus, these results show that açaí seed can be exploited as a source of polymeric proanthocyanidins.
The crude extracts were also analyzed using hydrophilic interaction chromatography (HILIC). A diol column separates PACs by size, allowing the report of results to be given in degree of polymerization (DP) after calibration [33]. A fluorescence detector (FLD) and mass spectrometry detector (micro-TOF) were used for comparison. Commercially available standards of epicatechin and procyanidin B2 were injected for retention time calibration for both methods (data not shown). Using the HILIC-HPLC-FLD analysis (Supplementary Material, Figure S1), PA, WA, and BRS crude extracts displayed individual peaks representing a degree of polymerization from 1 up to 12. The asymmetry in the peaks could indicate different PAC types in the samples or low column resolution [34].
The results obtained from HILIC-HPLC-FLD and HILIC-HPLC-DAD-ESI-TOF-MS are comparable. However, it also displays how the disparity between the results can arise, for instance, from analytical differences. Both methodologies showed that açaí seeds (E. oleracea Mart.) crude extracts are composed of oligomeric procyanidins, even though HILIC-HPLC-FLD indicated a higher degree of polymerization in the extracts.
The ethyl acetate fractions were analyzed by direct infusion ESI-MS/MS in negative mode (Table 2). PA and BRS displayed an m/z 289.2 ion, with MS 2 fragments with m/z 245 (a CO 2 loss) and m/z 205 (C 4 H 4 O 2 loss from the A-ring) ions indicative of (epi)catechin. An m/z 421.3 (289 + 132 Da) ion characteristic of a (epi)catechin-pentoside with a fragment of m/z 289.34 was found. Both samples exhibited an m/z 577.2 ion with MS 2 fragments of m/z 425 and m/z 407 (425-H 2 O) characteristic of a retro-Diels-Alder (RDA) fission of B-type procyanidin dimer. It also displayed a MS 2 fragment of m/z 451, a product of heterocyclic ring fission (HRF), and MS 2 fragments of m/z 287 and m/z 289, indicative of a quinone methide fission [35]. The WA EtOAc fraction exhibited the same ions and fragmentation pattern of PA and BRS varieties, except for an unidentified compound with m/z 469 ion and an MS 2 of m/z 289. This confirms that procyanidins are the main polyphenols in the açaí seeds, as corroborated by the literature [10][11][12]36].
As established by the liquid-liquid partitioning, the aqueous fractions are rich in polymeric PACs. Therefore, it was used for phloroglucinolysis of all varieties, and the results are shown in Table 1 (chromatograms are available in Supplementary Material, Figure S5). All varieties exhibited similar results, with a subunit composition profile of (−)-epicatechin as the single extension subunit found and a high percentage for (+)-catechin as a terminal subunit (above 80%). Their mean degree of polymerization (mDP) was also comparable, PA exhibited an mDP of 10.29, WA had an mDP of 11.23, while BRS aqueous fraction displayed an mDP of 11.81. Conversion yields for all the samples were above 80% which favors the homogeneity of the results. All açaí varieties presented a high polymerized B-type procyanidin. A previous study indicated similar results for açaí seed extracts with a high (−)-epicatechin content for extension subunit (above 95%), a high (+)-catechin percentage as terminal subunit (above 60%), and an mDP between 9.7-13 [11], corroborating that açaí seed extract might be a source of bioactive procyanidins, adding value to this by-product while decreasing the environmental impact.
MALDI-TOF mass spectra of PA, WA, and BRS aqueous fractions (Supplementary Material, Figure S6) showed clear repetitive patterns of peaks that allow the identification of specific oligomer series. Table 3  The BRS aqueous fraction also had the same pattern with a B-type procyanidin series (DP = 3-11), a procyanidin-prodelphinidin heterogeneous series (DP = 3-11), and a B-type procyanidin with one galloyl substitution (DP = 4-10). WA showed similar patterns with a B-type procyanidin (with DP = 3-11) and heterogeneous procyanidin-prodelphinidin sequences (with DP = 3-10). However, it did not have a galloyl substitution sequence. Heterogeneous sequences with procyanidin-prodelphinidin units in other plants are commonly reported in the literature [27,37]. All açaí varieties exhibited an arrangement with a one-unit exchange from procyanidin for prodelphinidin. Nevertheless, the pholoroglucinolysis reaction and MALDI-TOF data were consistent, showing that B-type procyanidins are the major components in the aqueous fractions of Euterpe spp. analyzed.
The E. oleracea Mart. seeds (PA, WA, and BRS varieties) are by-products with no primary exploration and with ecological implications due to accumulation. PA seed extract displayed a higher concentration of PACs (BuOH/HCl assay) and, therefore, was chosen for the corrosion experiments.
The PA crude extract corrosion inhibition activity was analyzed after 24 h of immersion in corrosive solution by fitting an LPR curve with ANOVA software to calculate the Tafel parameters, shown in Table 4, and by potentiodynamic curves (Figure 1). The PA crude extract only promoted an expressive effect at the concentration of 1.0 g/L, both increasing the corrosion potential (E corr ) and polarization resistance. The effect on E corr suggests an anodic type of corrosion inhibitor, but the increasing polarization resistance is typical for the cathodic type of corrosion inhibitor. Therefore, PA crude extract behavior suggests inhibition for both cathodic and anodic reactions [38,39].
The PA crude extract efficiency as a corrosion inhibitor was estimated by corrosion current density (J corr ) and by polarization resistance (Rp) of the metallic surface to evaluate the metal susceptibility to corrosion and the stability of the protective film on the metal surface, respectively. Table 4 shows that the anticorrosion activity occurred only at the concentration of 1.0 of PA crude extract. This data was corroborated by corrosion rate, also shown in Table 4. The efficiency of a corrosion inhibitor depends on a concentration range with an ideal number of molecules to form a stable, protective film. The absence or abundance of molecules leads to unstable film formation, either due to an insufficient number of molecules or the dispute of metallic surface interaction by excess of molecules [39].  The corrosion inhibitor efficiency of over 99.9% and a considerable reduction of corrosion rate shows that 1.0 g/L of PA crude extract, in neutral pH after 24 h of immersion, is a promising green corrosion inhibitor. Furthermore, the efficiency obtained for 1.0 g·L −1 of PA crude extract is higher than studies with PACs from other natural sources [25,26].
The Tafel slopes of 0.1 to 0.8 g/L and corrosion rate data showed a more intense corrosive process than that observed for the control (absence of inhibitor). These results prove that a weak corrosion inhibitor can promote the corrosive process [39,40]. However, the significant reduction on the anodic (β anodic ) and cathodic (β cathodic ) coefficients in the presence of 1.0 g/L 1 of PA crude extract shows the importance of the ideal concentration of inhibitor for inhibition of corrosion reactions.
The potentiodynamic curves (Figure 1) corroborated the suggestion that PA crude extracts affect anodic and cathodic reactions. After 24 h of immersion, in the condition of 1.0 g/L of PA crude extract, both anodic and cathodic branches showed low values of current density compared to the control. The other tested concentrations showed similar curves to those observed for the control.
Adsorption inhibitors affect the corrosion reaction in anodic and cathodic branches, as observed in the presence of 1.0 g/L of PA crude extract with a significant reduction of corrosion current density (Figure 1). This behavior is standard in organic compounds, such as PACs, for which there are already records in the literature [25][26][27]41]. The protective film was formed by adsorption of molecules on the metal surface, as the black/dark purple film on the metal surface in 1.0 g/L of PA crude extract was observed (Supplementary, Figure S7).
The effect of PACs on the metal surface was analyzed through EIS, displayed as Nyquist and Bode plots (Supplementary Material, Figures S8 and S9). The impedance consists of the relation between the alternating potential and the alternating current in the response of known frequency applications. Although the impedance is inversely proportional to the current, higher impedance modules promote lesser corrosion susceptibility. For the Nyquist plot in the presence of PA 1.0 g/L crude extract, the semicircle suggests the formation of a protective layer, blocking or reducing the effect of corroding molecules of the electrolyte on the metal surface. In the Bode plot with PA 1.0 g/L crude extract, there is a peak of the phase angles at low frequency, corroborating the protective film formation on the metallic surface hypothesis. Thus, as observed in the Nyquist plot, the presence of PACs inhibits the corrosion process. Furthermore, lower concentrations (0.2-0.8 g/L) probably do not have a sufficient number of molecules to form an efficient protective layer. However, in 1.0 g/L, the molecules content is ideal for metal surface adsorption, avoiding corrosive processes.
Previous studies have revealed açaí seeds as a promising source of antioxidants, with a higher antioxidant activity than the pulp bioactive compounds [7,11,12]. However, this is the first time that açaí seed extracts were tested for corrosion inhibition activity. In the PA crude extract, the high radical scavenging capacity and reducing metal activity of PACs translated to a promising green corrosion inhibitor for carbon steel AISI 1020 in neutral pH conditions at room temperature.
The protective activity can be affected by inhibitor chemical composition, metal type, and experimental physicochemical conditions. These variables can affect (positively or negatively) the interaction between the inhibitor and the metallic surface and consequently its anticorrosion activity [42][43][44]. Especially for PACs, their origin affects the polymerization degree, and it involves the interaction with the metal surface and the stability of the protective film. It was previously described that polymeric PACs are more effective for metal surface protection than monomeric ones [27,[42][43][44].
Crude extracts from açaí (E. oleracea Mart.) seeds and coconut (Cocos nucifera L.) husk fiber are natural sources of phenolic compounds. Comparing the polymeric degree of PACs from these natural sources showed that those PACs from açaí seed crude extracts had a higher polymeric degree than those from coconut husk fiber [25]. Furthermore, the PA crude extract proved to be more effective (higher inhibition efficiency and lower concentration) for carbon steel metal protection than coconut husk fiber crude extract. This supports the relation between high polymerization degree and corrosion inhibition activity.

Extraction Procedures
E. oleracea Mart. seeds from purple açaí (PA) and white açaí (WA) were obtained in São João de Pirabas (Pará, Brazil) by a local private producer. The E. oleracea Mart. BRS-Pará (BRS) seeds were graciously donated by Embrapa Oriental Amazonia (Belém, Pará). The seeds had their pulps removed and were washed, air-dried, and transported to the laboratory. The seeds were grounded, defatted (20.0 g, triplicate) by Soxhlet extraction with n-hexane (3 × 4 h). The defatted powdered seeds were extracted (2.0 g, triplicate) with Me 2 CO:H 2 O 6:4 in an ultrasound bath for three 10 min cycles, renewing solvent in each period. The crude extracts were vacuum filtered; the acetone was evaporated at 35 • C in a rotatory evaporator and then lyophilized.

Ethyl Acetate:Water Liquid-Liquid Partitioning
Crude extracts were dissolved in 50 mL of water and then partitioned with the same volume of ethyl acetate three times. The organic phase was evaporated in a rotatory evaporator, and the aqueous fraction was lyophilized.

Proanthocyanidin Content by n-BuOH/HCl Test
The determination of proanthocyanidin content followed a published protocol [30]. The grounded and defatted seeds (0.2 g in triplicate) were submitted to ultrasound-assisted extraction with acetone:water 7:3 (10 mL) for 10 min. A 5 µL aliquot of extract was added to a sealed test tube containing 3 mL of n-butanol/hydrochloric acid (95:5) and 100 µL of a 2% solution of NH 4 Fe(SO 4 ) 2 in 2N hydrochloric acid. The solution was heated for 60 min (95 • C). After cooling, the absorbance was measured at 550 nm (UV-1601 Shimadzu spectrophotometer). Results were expressed in percentage (%) by dry matter.

MALDI-TOF-MS
The mass spectrometry analyses were made on a MALDI-TOF Autoflex Speed Bruker spectrometer using a published protocol [35] with slight modifications. Samples (2 mg) were dissolved with 0.1% aqueous trifluoroacetic acid and diluted to 1:10 with DHB matrix solution (0.1% TFA). A 1.0 µL aliquot of aqueous sodium chloride (1.0 mg/mL) was added to the solution. The samples were applied to the MALDI plate (duplicate) and analyzed after they were dried. Analyses were made in positive mode, using a peptide calibration standard (Bruker, Billerica, MA, USA). Data were processed using mMass 5.5.0 software (Strohalm M. ® ).

Phloroglucinolysis
Phloroglucinolysis determinations used a previously published protocol [45] with a phloroglucinol solution prepared with 5.0 g of phloroglucinol, 1.0 g of ascorbic acid, and 0.1 N HCl in a minimum amount of methanol up to 100 mL. NaHCO 3 solution was prepared with 336.0 mg of NaHCO 3 and Milli-Q water up to 100 mL (40 mM).
Capped glass test tubes containing 5.0 mg weighted samples (triplicate) with 1.0 mL of the phloroglucinol solution were heated for 20 min in a water bath (50 • C). An aliquot of 200 µL was transferred to a 2.0 mL vial, and 1.0 mL of the NaHCO 3 solution was added. A ReproSil-Pur RP-18 column (250 × 4.6 mm, 5 µm, Dr. Maisch GmbH, Germany) with a guard column of the same material was used; flow rate: 1 mL/min, 280 nm UV detector. Mobile phase (A) was aqueous 1% acetic acid, and (B) was methanol. Gradient mode: 5% B for 10 min, 5-20% B in 20 min, 20-40% B in 25 min, 90% B for 10 min, 5% B for 5 min.

Corrosion Experiments
The corrosion tests were carried out in a three-electrode (a working electrode, a reference KCl-saturated calomel electrode (SCE), and a platinum counter electrode) electrochemical cell which was connected to an AUTOLAB PGSTAT302N potentiostat/galvanostat. Square coupons of carbon steel AISI 1020 (Fe-98.5%, C-0.2%, Mn-0.6% and traces of P, S, Si, Sn, Cu, Ni, Cr, and Mo) were used as working electrodes. The corrosive solution simulates cooling systems water and was composed of 500 ppm chloride (829 mg/L NaCl), 150 ppm of sulfate (222 mg/L Na 2 SO 4 ), and 150 ppm of calcium carbonate (CaCO 3 ). The pH electrolyte was controlled in neutral range (7.0 ± 0.2) during all immersion times with different concentrations of PA crude extract (0-1.0 g/L).
These tests were conducted in 800 mL glass cells, maintained at controlled temperature (25 ± 2 • C), and slow shaken for 24 h for data acquisition. Before each test, carbon steel AISI 1020 coupons (working electrodes), with 1.0 cm 2 of the exposed area, were mechanically polished using SiC papers of 80, 120, 220, 320, 400, 500 and 600 grit, washed with distilled water, cleaned with ethanol and dried in hot air. And, the lyophilized PA crude extract was resuspended in distilled water and sterilized by membrane filtration (0.22 mm pore) to prepare a stock solution, used as basis for the tests PA crude extract solutions.
The activity as corrosion inhibitor was determined through the comparison between the results in absence and presence of different concentrations of PACs in PA crude extract and performed using LPR and potentiodynamic curves techniques. The concentrations tested were 0.1, 0.2, 0.5, 0.8 and 1.0 g/L. The potentiodynamic polarization curves were performed by working electrode scanning from −800 to −500 mVSCE at a scan rate of 0.33 mVs −1 . And the Tafel data acquisition applied linear polarization resistance (LPR) technique by working electrode scanning from −20 to 20 mVSCE around the corrosion potential at a scan rate of 0.33 mVs −1 . The inhibition efficiencies were calculated trough two data. The first method applied Tafel data (Equation (1)), where J corr and J 0 corr are the corrosion current density values with and without inhibitor, respectively. The second one by linear polarization resistance (LPR) data (Equation (2)), where R p and R 0 p are the resistance polarisation values in the presence and absence of inhibitor.

Conclusions
In this study, all analytical techniques determined that the three açaí varieties (PA, WA and BRS-Pará) are a potential source of PACs with a high degree of polymerization, which could have industrial applications. All varieties exhibited a similar chemical composition, indicating that the seeds do not need to be separated to exploit their phenolics. Furthermore, PA crude extract showed itself as a promising green corrosion inhibitor for carbon steel AI-SI 1020 in neutral pH corrosive solutions at room temperature. A black/dark purple film was observed on carbon steel AISI 1020 surface after 24 h of immersion in the presence of 1.0 g/L of PA crude extract, suggesting the inhibitor adsorption and a protective film formation. The inhibition efficiency of over 99.9% indicated the açaí seed extract is a promising target for future studies about corrosion inhibitors from natural sources, especially from a raw solid waste as the açaí seeds.
Supplementary Materials: Figure S1-HILIC-HPLC-FLD chromatograms of PA, WA and BRS crude extracts. Peaks are identified by their degree of polymerization (DP). Figure S2-HPLC-DAD chromatogram of PA extract. Extracted ion chromatograms corresponding to B-type procyanidins oligomers (monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer and octamer, respectively). Figure S3-HPLC-DAD chromatogram of WA extract. Extracted ion chromatograms corresponding to B-type procyanidins oligomers (monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer and octamer, respectively). Figure S4-HPLC-DAD chromatogram of BRS extract. Extracted ion chromatograms corresponding to B-type procyanidins oligomers (monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer and octamer, respectively). Figure S5-Chromatograms of PA, WA and BRS phloroglucinolysis aqueous fractions. Figure S6-MALDI-TOF [M+Na]+ mass spectra from PA, WA and BRS aqueous fractions. Figure S7-Effect of PA on the metallic surface after 24 h immersion in neutral pH corrosive solution without PA and 1.0 g·L −1 of PA. A-Metallic surface after 24 h immersion in neutral pH corrosive solution; B-Black protective film formation after 24 h immersion in neutral pH corrosive solution with 1.0 g·L −1 of PA; and C-metallic surface after black film removal. Figure S8-Nyquist plot for carbon steel AISI 1020 in a neutral pH corrosive solution for purple açaí (PA) crude extract in 1.0 g·L −1 . The control represents the absence of any inhibitor. Figure S9-Bode plot for carbon steel AISI 1020 in a neutral pH corrosive solution purple açaí (PA) crude extract in 1.0 g·L −1 . The control represents the absence of any inhibitor. Relation between log frequency (Hz) and phase angle ( • ) and log impedance modules (Ω·cm −2 ).