pharmaceutics Article Purification and Identification of Novel Antioxidant Peptides Isolated from Geoffroea decorticans Seeds with Anticoagulant Activity Juliana Cotabarren 1, *, Brenda Ozón 1 , Santiago Claver 1 , Javier Garcia-Pardo 2, * and Walter David Obregón 1, * 1 Centro de Investigación de Proteínas Vegetales (CIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de la Plata, Buenos Aires B1900, Argentina; brendaozon@gmail.com (B.O.); claversantiago14@gmail.com (S.C.) 2 Institut de Biotecnologia i Biomedicina and Departament de Bioquimica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain * Correspondence: cotabarren.juliana@biol.unlp.edu.ar (J.C.); javiergarciapardo@msn.com (J.G.-P.); davidobregon@biol.unlp.edu.ar (W.D.O.); Tel.: +54-221-423-5333 (J.C. & W.D.O.); +34-93-581-1233 (J.G.-P.) Abstract: Geoffroea decorticans is a xerophilous deciduous tree present in most arid forests of southern South America, which is commonly used in traditional medicine. The seeds of this tree have been previously investigated for their singular chemical composition, but their protein content has been poorly investigated. Herein, we report the isolation, purification, and characterization of a set of thermostable peptides derived from Geoffroea decorticans seeds (GdAPs) with strong antioxidant and anticoagulant activities. The most potent antioxidant peptides showed a half maximal inhibitory   concentration (IC50 ) of 35.5 ± 0.3 µg/mL determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH). They also caused a dose-dependent prolongation of the aPTT clotting time with an IC50 value of Citation: Cotabarren, J.; Ozón, B.; Claver, S.; Garcia-Pardo, J.; Obregón, ~82 µg/mL. Interestingly, MALDI-TOF/MS analysis showed the presence of three major peptides W.D. Purification and Identification of with low molecular weights of 2257.199 Da, 2717.165 Da, and 5422.002 Da. The derived amino-acid Novel Antioxidant Peptides Isolated sequence of GdAPs revealed their unique structural features, exhibiting homology with various from Geoffroea decorticans Seeds with proteins present in the genome of Arachis hypogaea. All in all, our data suggest a direct applicability Anticoagulant Activity. Pharmaceutics of GdAPs for pharmaceutical purposes. 2021, 13, 1153. https://doi.org/ 10.3390/pharmaceutics13081153 Keywords: Geoffroea decorticans; antioxidant; functional compound; thermostable protein; reactive oxygen species; anticoagulant; bioactive compound Academic Editor: Maria Rosaria Lauro Received: 14 June 2021 1. Introduction Accepted: 22 July 2021 Published: 27 July 2021 Over many centuries, plants have historically made a major contribution to phar- macology. In fact, an important number of currently available drugs are derived directly Publisher’s Note: MDPI stays neutral or indirectly from natural products, especially as antibacterial or antitumor agents [1,2]. with regard to jurisdictional claims in However, there is growing evidence that modern pharmacology has only explored and vali- published maps and institutional affil- dated a small part of this large array of molecular entities. The best-characterized examples iations. of bioactive molecules derived from plants include peptides, proteins, sugars, nucleo- sides, or products of the secondary metabolism of the plant. Among them, plant-derived peptides have shown a variety of interesting biological activities such as hypertensive, hy- poglycemic, antioxidant, and/or anticoagulant properties. This broad bioactivity spectrum Copyright: © 2021 by the authors. of these peptide molecules makes them attractive for the treatment or prevention of various Licensee MDPI, Basel, Switzerland. diseases [3–5]. This article is an open access article In recent years, substantial research efforts were dedicated toward the discovery of distributed under the terms and novel drugs to treat cardiovascular diseases (CVDs). CVDs are a group of disorders of the conditions of the Creative Commons heart and blood vessels that represent one of the major causes of mortality worldwide. In Attribution (CC BY) license (https:// most cases, the drugs used to treat CVDs are anticoagulant molecules that inhibit or delay creativecommons.org/licenses/by/ coagulation by acting on one or more coagulation factors. Coagulation and fibrinolysis are 4.0/). interrelated enzymatic processes that, together with mechanisms involving the vascular Pharmaceutics 2021, 13, 1153. https://doi.org/10.3390/pharmaceutics13081153 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 1153 2 of 12 wall and platelets, keep the blood flowing within the blood vessels. An inadequate activa- tion of coagulation or the uncontrolled activation of fibrinolysis can generate hemorrhagic disorders, such as in Von Willebrand’s disease, hemophilia, or hemolytic anemia [6]. It is known that a cascade of enzyme activations occurs in the coagulation process, which are mainly regulated by inhibitors of a protein nature [7]. Current pharmacological agents used to prevent or treat thrombotic disorders are mostly synthetic antiplatelet, anticoag- ulants, and fibrinolytic agents [8], which remain under continuous review due to their adverse effects and bleeding risks [9,10]. Thus, the identification of novel anticoagulant compounds from natural sources has gained interest because of their direct application in the treatment of CVDs and other associated diseases such as viral infections [11], can- cer [12], malaria [13], hypertension [14,15], and/or blood disorders [16]. On the other hand, it has been previously demonstrated that reactive oxygen species (ROS) play a vital role in the development and progression of CVDs [17–21]. In fact, generation of ROS and free radicals have deleterious effects on many molecules in the human body, such as DNA, fatty acids, and proteins [22]. Typically, proteins and peptides can inhibit oxidative reactions, in part due to their ability in neutralizing the effects of transition metals, leading to the formation of less reactive insoluble metal complexes [23]. Recent studies have shown that crude proteins from some of the seeds of plant species such as pea [24], chickpea [25], and peanut kernels [26] display strong antioxidant activity associated with a low toxicity profiles [27]. A growing number of bioactive peptides have been identified from various medicinal plant sources [28–31]. Geoffroea decorticans (Gill. ex Hook. et Arn.) Burkart (Fabaceae) is a xerophilous deciduous tree mostly found in arid areas of South America used in traditional medicine. This plant is also known commonly as chañar. Chañar fruits have been con- sumed fresh or cooked by human communities since ancient times [32,33]. The fruits of G. decorticans are brown spherical drupes with sweet pulp and palatable taste. The edible part is composed of the fleshy mesocarp that, air-dried, contains 11.10% sugars and 4.49% resins with 8.75% protein, 15.20% reducing sugar, and 52.89% starch and other carbohydrates. Chañar seeds are also edible (both fresh or roasted) with about 22% protein and a high oil content of about 47%, where the oleic–linoleic ratio stands out (53.7%:30.7%) [34]. In addition, some studies have reported antifungal properties in the organic extracts of the chañar stem bark, especially associated with inflammatory processes [35–38]. Despite all these previous studies, the protein composition and activity of the bioactive compounds present in this plant have been poorly characterized. Herein, we report the isolation and characterization of a set of thermostable bioactive peptides obtained from G. decorticans seeds. These native peptides showed a potent antioxidant activity in vitro. Furthermore, we demonstrate the suitability of these bioactive peptides as anticoagulant molecules targeting the intrinsic pathway of coagulation. Taken together, the current results provide useful information for the production of novel plant- derived anticoagulant molecules with additional functionalities. 2. Materials and Methods 2.1. Materials 2.1.1. Chemicals Coomassie Blue G-250, sodium chloride, tris(hydroxymethyl) aminomethane, sodium dodecyl sulfate (SDS), N,N,N 0 ,N 0 -tetramethylethylenediamine (TEMED), β-mercaptoethanol (βME), 1,1-diphenyl-2-picrylhydrazyl (DPPH), bovine serum albumin (BSA) and 2,20 -azino- bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Glyoxyl-agarose was purchased from FlukaTM (Charlotte, NC, USA). 2.1.2. Sample Collection The ripe fruits of Geoffroea decorticans were hand-collected from 20 trees located in the department of Juan Martín de Pueyrredón (33◦ 160 02.900 S 66◦ 120 50.200 W), Juana Koslay, Pharmaceutics 2021, 13, 1153 3 of 12 San Luis province, Argentina. The samples were collected in January 2018 and identified as Geoffroea decorticans (Gill., ex Hook & Arn, Burkart, family Fabacea) by specialists of the Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Argentina. A voucher specimen was deposited at the Herbarium of the Universidad Nacional de San Luis (UNSL Del Vitto N◦ 553). G. decorticans fruits were flash-frozen and stored at −20 ◦ C until use. 2.2. Crude Extract Preparation and Determination of the Protein Content Chañar seeds were obtained from G. decorticans (chañar) fruits after the woody en- docarp was hand-broken and removed. To prepare the crude extract, 10 g of seeds were washed with distilled water. The sample was ground using a blender in 100 mL of 0.1 M Tris–HCl buffer, pH 7.5. This process was performed on an ice bath to reduce protein denaturation. After incubation for 120 min at 4 ◦ C, the mixture was filtered through a fine screen cloth and centrifuged for 30 min at 7000× g at 4 ◦ C. Afterward, the supernatant (hereafter GdCE) was collected and immediately flash-frozen at −20 ◦ C. The soluble protein was determined using Bradford’s assay [39]. Briefly, to perform the reactions, 10 µL of each standard solution or test sample was mixed with 200 µL of the Bradford reagent. After 10 min incubation at room temperature, the absorbance at 595 nm was recorded using a Tecan Infinite M200 PRO spectrophotometer (Männedorf, Switzerland). Bovine serum albumin (BSA) was used as a protein concentration standard. This method is particularly suitable for the determination of protein content of vegetable extracts, which usually have phenolic compounds that interfere with the traditional Lowry method. All the measurements of the soluble protein content were carried out in triplicate. 2.3. Purification of Antioxidant Peptides 2.3.1. Initial Purification by Heat Treatment The crude extract (GdCE) was subjected to a water bath at 90 ◦ C for 30 min. After cooling the sample, precipitated proteins were removed by centrifugation at 4 ◦ C for 30 min at 7000× g. After centrifugation, the soluble protein content and the antioxidant activity of the nontreated crude extract and the heat-treated sample (GdHT) were determined. SDS- PAGE analysis was used to determine the protein profile during the different purification steps. Gels were silver-stained following the Chevallet, Luche, and Rabilloud [40] protocol. Densitograms for each lane were produced using the GelAnalyzer 19.1 program. The obtained peaks were compared according to computed Rf (Retardation factor) values and band intensity, which is proportional to the protein concentration on each peak. 2.3.2. Size-Exclusion Chromatography A 1 mL aliquot of the heat-treated sample (1767 µg/mL) was loaded onto a Sephacryl- S100 HR column (1.5 × 40 cm) connected to an Äkta-Purifier (GE Healthcare) previously equilibrated with milliQ water. Elution was carried out using an isocratic gradient and a flow rate of 0.8 mL/min. During elution, the absorbance was continuously monitored at 280 and 215 nm. The antioxidant activity was determined for each fraction, and the fraction with the higher antioxidant activity was selected for further analysis. 2.4. MALDI-TOF Mass Spectrometric Analysis To perform mass spectrometry (MS) analysis, an aliquot of the sample from the exclusion chromatography was desalted by dialysis in 0.022 µm filters. The dialyzed sample was mixed with an equal volume of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) matrix solution. Then, the matrix–sample mixture was spotted onto an MTP 384 target plate (polished steel TF, Bruker Daltonics, Billerica, MA, USA). After the spots were evaporated to dryness at room temperature, the mass spectra were acquired on a Bruker Daltonics Ultraflex MALDI-TOF mass spectrometer (Bruker Daltonics) operating in linear positive and reflector mode. Pharmaceutics 2021, 13, 1153 4 of 12 The PMF analysis was performed for identification of the antioxidant peptides of Chañar. First, an in situ tryptic digestion of the Tris–tricine SDS-PAGE electrophoresis lane containing the protein bands was performed by following a standard PMF protocol [41]. In brief, the tryptic peptides were dissolved in 10 µL of 0.1% CF3CO2H (v/v) and analyzed by MALDI-TOF in the presence of an HCCA matrix. Mass spectra of the tryptic peptides were acquired using a Bruker Daltonics Ultraflex MALDI-TOF mass spectrometer (Bruker Daltonics) in a reflectron positive mode operating at 25 kV acceleration voltage. In both cases, a standard peptide calibration mixture from Bruker Daltonics used as reference. Four precursor ions MH+ (1267.672, 1682.694, 1925.806, and 2778.353) were subjected to TOF–TOF fragmentation. The resultant fragmentation spectra for these precursor MH+ ions were compared with all the fragmentation patterns for all the sequences present in the MASCOT database, considering the carbamidomethylation of all Cys (www.matrixscience. com, access on 1 April 2020). All the MS experiments were performed at the Proteomics Core Facility CEQUIBIEM, at the University of Buenos Aires/CONICET (National Research Council). 2.5. Antioxidant Activity Evaluation 2.5.1. DPPH Radical-Scavenging Activity The Brand-Williams method was used to evaluate the DPPH scavenging activity against free radicals [42]. The method was adapted to a 96-well flat-bottom plate. In essence, 50 µL of each sample were mixed with 50 µL of a 0.2 mmol/L DPPH radical solution and 150 µL of methanol. After incubation for 20 min at 37 ◦ C in dark conditions, the absorbance of each well was measured at 517 nm using a microplate spectrophotometer. As a control condition, the maximum DPPH absorbance was determined by replacing the sample volume with the same volume of water. For IC50 determination, six different concentrations for each sample were analyzed. Measurements were carried out in triplicate. The IC50 values were calculated using GraphPad software. DPPH radical-scavenging activity was calculated using the following equation: A1 − A2 DPPH radical scavenging activity (%) = 100 × , A1 where A1 is the absorbance of the control without sample and A2 is the absorbance in presence of sample and DPPH. 2.5.2. ABTS Radical-Scavenging Activity The ABTS+ radical-scavenging activity was determined using the method described by Pukalskas et al. [43], with slight modifications. The antioxidant activity in the samples was analyzed by adding 10 µL of sample to 190 µL of diluted ABTS+ solution. After 10 min incubation at room temperature in the darkness, absorbance at 734 nm was measured using a microplate spectrophotometer. Measurements were carried out in triplicate. For IC50 determination, six different concentrations of each sample were analyzed. All the assays were carried out in triplicate. The IC50 values were calculated using GraphPad software. The ABTS+ radical-scavenging activity was calculated using the following equation: A1 − A2 ABTS radical scavenging activity (%) = 100 − 100 × , A0 where A0 is the absorbance of the control without sample and A1 is the absorbance in the presence of the sample and ABTS+ . A2 corresponds to the value of absorbance of sample blank without ABTS+ . 2.6. Anticoagulant Activity The anticoagulant activity of GdAPs was evaluated as previously described by our group [44]. In essence, we measured the prothrombin time (PT) and the time of activated partial thromboplastin (aPTT) using a Coatron M1 coagulometer (TECO, Germany). Before Pharmaceutics 2021, 13, x FOR PEER REVIEW 5 of 13 Pharmaceutics 2021, 13, 1153 The anticoagulant activity of GdAPs was evaluated as previously described 5by our of 12 group [44]. In essence, we measured the prothrombin time (PT) and the time of activated partial thromboplastin (aPTT) using a Coatron M1 coagulometer (TECO, Germany). Be- fore performing the experiments, a pool of blood plasmas obtained from five different performing samples ofthe experiments, healthy a pool individuals of blood was plasmas prepared, obtained aliquoted, from and five at stored different samples 37 °C with 3.8% ofsodium healthycitrate individuals was prepared, aliquoted, and stored at 37 ◦ C with 3.8% sodium (sample–anticoagulant ratio of 9:1). This reference sample was used in both citrate (sample–anticoagulant determinations ratio (hereafter PBP) toof 9:1). This evaluate thereference sample PT and aPTT wasFor time. used theinPTboth test,determi- the com- nations (hereafter PBP) to evaluate the PT and aPTT time. For the PT test, the commercial mercial Soluplastin reactive was used (Wiener Lab.) To perform reactions, equal parts of Soluplastin reactive was used (Wiener Lab.) To perform reactions, equal parts of the sample the sample PBP and GdAPs (0–215 µg/mL) were incubated for 2◦ min at 37 °C. Afterward, PBP and GdAPs (0–215 µg/mL) were incubated for 2 min at 37 C. Afterward, 50 µL of 50 µL of Soluplastin were added to 25 µL of the mixture and the sample was evaluated in Soluplastin were added to 25 µL of the mixture and the sample was evaluated in order to order to determine the coagulation time. For the aPTT test, 25 µL of aPTT (Wiener Lab.) determine the coagulation time. For the aPTT test, 25 µL of aPTT (Wiener Lab.) was added was added to an equal volume of PBP–GdAPs mixture (previously incubated for 2 min at to an equal volume of PBP–GdAPs mixture (previously incubated for 2 min at 37 ◦ C). After 37 °C). After incubation for 2 min at 37 °C, 25 µL of 50 mM CaCl2 was added to the sam- incubation for 2 min at 37 ◦ C, 25 µL of 50 mM CaCl2 was added to the samples. All the ples. All the experiments were performed at least in triplicate. experiments were performed at least in triplicate. 2.7.Statistical 2.7. StatisticalAnalysis Analysis Statisticalanalyses Statistical analyses(ANOVA) (ANOVA) were were performed performed using using GraphPad GraphPadPrism Prismv.v.01 01 software soft- (http://www.graphpad.com/scientific-software/prism/, access on 10 June ware (http://www.graphpad.com/scientific-software/prism/, access on 10 June 2021). 2021). A Tukey’s A post hoc Tukey’s testhoc post (p 215(IC50µg/mL). As a comparison, > 215 µg/mL). As a comparison,heparin, a drug commonly heparin, a drug commonlyused in used clinical in applications, in a concentration of 2 mg · mL −1 , produces a delay in coagulation time for clinical applications, in a concentration of 2 mg·mL , produces a delay in coagulation time −1 the the for aPTT route aPTT fromfrom route 18 to18300 to−s1300 [61], whilewhile s [61], GdAPs produces GdAPs the same produces effect effect the same at much lower at much concentrations of 172 µg · mL . This behavior was previously lower concentrations of 172 µg·mL . This behavior was previously reported for other pep- −1 reported for other peptides from from tides plant plant originorigin such as suchtheastrypsin inhibitors the trypsin from Leucaena inhibitors leucocephala from Leucaena [62], Enterolobium leucocephala [62], Ente- contortisiliquum [63], and Maclura pomifera [64]. rolobium contortisiliquum [63], and Maclura pomifera [64]. Figure 5. Anticoagulant activity of GdAPs on activated partial thromboplastin (aPTT) and prothrom- bin (PT). (A,B) Effect of GdAPs concentration on the clotting time and (B) activation rate of aPTT and PT. The vertical bars correspond to the standard deviation of three independent measurements (n = 3). In summary, the presented results show that GdAPs have significant beneficial effects as an anticoagulant drug. In addition, our results demonstrate that these peptides target the intrinsic pathway, leading to a retardation of the clotting time. Taken together, our results encourage future studies on the mechanism of action of GdAPs and the development of in vivo assays that could be promising for a potential exploitation of these peptides as naturally occurring antioxidant and antithrombotic agents. 4. Conclusions Plants represent a rich source of bioactive compounds suitable for pharmaceutical applications. Although G. decorticans is a native plant endemic of South America that is Pharmaceutics 2021, 13, 1153 10 of 12 recognized for its medicinal properties, only few reports of bioactive molecules isolated from this plant source are available so far [44]. In this study, we successfully isolated a set of native peptides present in G. decorticans seeds. To achieve this, the seed extracts were subjected to simple purification steps, such as heat treatment and molecular exclusion chromatography. Other previous reports used enzymatic hydrolysis with proteases as biocatalysts, which requires multiple time-consuming steps and additional reagents for the production of the bioactive peptides. Interestingly enough, these peptides have remarkable thermal stability, which is an added benefit of their conservation without the need for refrigeration. This would allow their use to be adapted to many geographical regions of the world that have a shortage of technologies and adequate infrastructure for storage. Lastly, we demonstrated that the peptides present in G. decorticans have a strong antioxidant capacity, comparable to other previously isolated molecules (see Table 2). More interestingly, we revealed that such molecules have a strong anticoagulant effect against the intrinsic coagulation pathway. All in all, our findings position G. decorticans as an attractive source of bioactive molecules and suggest a possible biomedical application of GdAPs isolated from the seeds of this plant. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics13081153/s1, Table S1: Protein sequences obtained from the NCBI database after performing an sequence similarity search for the peptides (b), (c) and (d). Author Contributions: J.C., B.O., S.C., and W.D.O. performed the experiments, analyzed the data, and wrote the manuscript. J.C., J.G.-P., and W.D.O. assisted with the design of experiments and with the writing and revision of the manuscript. All authors read and agreed to the published version of the manuscript. Funding: J.C. is a postdoctoral fellow from the Argentine Council of Scientific and Technical Research (CONICET), B. Ozón is a doctoral fellow from the National Agency of Scientific and Technological Promotion (ANPCyT-Argentina), and W.D.O. is member of the Researcher Career Program of the CONICET. The authors acknowledge the support from Universidad Nacional de La Plata (Argentina) under grants PPID X/014 and PPID X/038, the UNLP I+D 2020 project Code X-920, and the National Agency of Scientific and Technological Promotion (Argentina) under Project PICT-2016-4365 and PICT-2018-03271. Institutional Review Board Statement: Not applicable. 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