Extracts with protective effect against lipid oxidation of edible oils were obtained from papaya (Carica papaya L.) seeds through supercritical fluid extraction (SFE). SFE was performed with pure carbon dioxide (CO2) and CO2 modified by ethanol (ETOH) at different temperatures (40, 50, 60 0C) and pressures (10, 20, and 30 MPa). The protective effect against lipid oxidation was evaluated in vegetable edible oil (EO) and compared with the synthetic antioxidant butyl hydroxytoluene (BHT). For comparison, Soxhlet extraction (SE) was performed with n-hexane and EtOH. The extracts with highest protective effect were analyzed by high performance liquid chromatography with diode array detection (HPLC-DAD), and some phenolic acids (gallic, p-hydroxybenzoic, caffeic, p-coumaric and ferulic) and flavonols (quercetin and kaemferol) were identified. The highest extraction yields resulted in SE with EtOH (31.46 Â± 0.10%) and by SFE with CO2/EtOH (23.75 Â± 0.04%). The extracts obtained by SE with EtOH, by SFE with CO2 at 20 MPa (40, 50 and 60 °C) and with CO2/EtOH (20 MPa, 50°C and 5% ETOH) have shown the highest protective effect on EO, higher than that observed for BHT. Hence, papaya seeds are a promissory source of natural antioxidants for vegetable edible oils, which might replace synthetic industrial products.
Keywords: Carica papaya L. seeds, lipid oxidation, supercritical fluid extraction.
Papaya (C. papaya L.) is a native fruit of tropical America. Its global production in 2012 was about 3.52 million tons, with India and Brazil the major producers with 1.46 and 0.43 million tons, respectively, whereas Colombia produced about 46500 tons . The fruit is grown mainly for fresh consumption, as well as for juice and papain production. The international market shows many processed products, discarding the seeds, these can reach 15% of the fruit . In traditional medicine the papaya seeds have been used against malaria, as sedative, muscle relaxant and as anticonvulsant . Various biological effects of papaya seed extracts have been reported: ethanolic extracts showed antibacterial activity, as well as ovicidal and larvicidal activity , aqueous extracts showed anthelmintic and antiamoebic activity [4,5], whereas methanolic extracts exhibited a slight antiinflammatory activity in rats . The contraceptive effect of papaya seed extracts is well documented; polar extracts possess a contraceptive efficacy in rats , monkeys , rabbits  and dogs . In recent times, an antifertility compound was isolated from it . By other way, recently the benzylglucosinolate was isolated and identified from papaya seeds, this compound showed a high efficiency against lipid oxidation in edible oils (EO) .
EO submitted to heating and storage suffer lipid oxidation, leading to deterioration of sensory and nutritional properties, and formation of toxic compounds. Synthetic antioxidants like BHT and TBHQ have been widely used to delay the oxidation process, however, these have been questioned for its adverse effect on consumer health [13,14]. The actual trend is to use natural sources of antioxidants in food conservation, the papaya seeds would be one of those sources.
Supercritical fluid extraction (SFE) is an alternative technique, by which antioxidant extracts can be obtained; various studies have shown the effectiveness of SFE in obtaining antioxidants with ability to retard the lipid oxidation in foods [15-17]. Our research group has explored SFE of fruit wastes and obtained extracts with AA in food like cooked beef meat , canola oil  and linoleic acid emulsion . The results obtained have shown that SFE is an efficient and selective method to obtain antioxidants with protective effect in foods. For papaya seeds yet there are no reports about the use of supercritical fluid extraction to obtain antioxidant extracts.
This work explores the papaya seeds as a source of antioxidants capable to delay the lipid oxidation in vegetable EO. Therefore, different extracts were obtained employing SFE with carbon dioxide (CO2) and with CO2 modified by ethanol (EtOH) as a co-solvent (CO2/EtOH). The results from SFE were compared to results from Soxhlet extraction (SE) with EtOH as solvent, in terms of extraction yield and protector effect on EO. In the extracts with highest AA some phenolic compounds were identified by HPLC-DAD.
Samples, chemicals and standards
Alimentos SAS S.A. (Bogotá-Colombia) provided the papaya seeds, these being for them a waste product from papaya fruit processing. A refined bleached and deodorized vegetable EO without antioxidants, was supplied by Duquesa S.A. (Bogotá-Colombia). This was a mixture of palm, soy and sunflower oils, composed of 70% of triglycerides (TG) with unsaturated fatty acids (60% oleic, 35% linoleic acid and 5% others) and 30% of TG with stearic acid. Papaya seeds and the EO were stored in dark at -20 and -80 °C, respectively, until use.
CO2 (99.9% purity) was purchased from White Martins Praxair Inc (Joinville-SC, Brazil). Ethanol (EtOH), isooctane, trichloro-acetic acid (TCA) and chlorhydric acid (HCl) were purchased from Vetec Química Fina Ltda (Rio de Janeiro-RJ, Brazil). Thiobarbituric acid (TBA), hexanal (HEX) and nonanal (NON) were from Alfa Aesar (Lancashire, UK). Ferrous chloride dihydrate was purchased from Merck (Darmstadt, Germany). Butylated hydroxytoluene (BHT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis-MO, USA). Chloroform and hexane were obtained from Synth (Diadema-SP, Brazil). Solid-phase micro-extraction fibers were purchased from Supelco Sigma-Aldrich (St. Louis-MO, USA). All used reagents and solvents were either analytical or HPLC grade.
The papaya seeds were cleaned using running water. Subsequently they were dried at room temperature for 72 h until a final moisture content of 0.3%. The dried seeds were crushed using a knife mill (De Leo, Porto Alegre-RS, Brazil) and then the particles were separated by size in a vertical vibratory sieve shaker (Bertel Metalurgic Ind. Ltda., Caieiras-SP, Brazil). The resulting seed material with particle size between 0.300 and 0.850 mm (+50/-18 US Standard size sieves) was used in all extractions, the mean particle diameter was 0.496 Â± 0.003 mm calculated based on the mean size distribution . The dry and grounded samples were stored in plastic bags at -20 ºC.
The SFE of papaya seeds was performed in a dynamic extraction unit [22,23]. The equipment contains a pressurized CO2 reservoir, a cold bath (Thermo Haake C10, Karlsruhe, Germany) kept at -10 °C in order to keep the CO2 in liquid state, an air driven pump (Maximator M111, Walkenrieder StraÃe Zorge, Germany), a stainless steel jacketed extraction column (329 mm length, 20.42 mm inner diameter and internal volume of 100 mL), pass valves, flow regulators and manometers. The extraction temperature was controlled by a thermostatic bath (Thermo Haake DC30 Karlsruhe, Germany). A co-solvent pump (Constametric 3200, Thermo Separation Process, Riviera Beach-FL, USA) was connected to the extraction line in order to supply the modifier (organic solvent at high-pressure) at a pre-established flow rate, to mix into the CO2 flow before the extraction vessel. The extraction procedure was previously established . A sample of papaya seeds (10 g) was placed inside the extraction column as a fixed bed of particles, and the process variables (temperature, pressure and solvent flow rate) were adjusted to give the desired continuous flow. The mixture supercritical solvent+extract was released to ambient pressure using a pressure regulator valve and the extracts were collected in amber flasks. Each extraction was performed for 180 min (according to previous kinetics assays), finally the extracts were weighted in an analytical balance (SHIMADZU Model AY220, SÃ£o Paulo-SP, Brazil), then flushed with a nitrogen stream, sealed, and stored at -20 °C.
The SFE assays were divided into two groups:
Pure CO2 assays using the supercritical CO2 at different temperatures (40, 50 and 60 ºC), and different pressures (10, 20 and 30 MPa), at a CO2 flow rate of 0.50 Â± 0.05 kg/h. The extraction temperatures and pressures were selected based on previous experience and the maximum operational conditions of the extraction unit.
Assays with CO2/EtOH, where the EtOH was added to supercritical CO2 in concentrations of 2, 5 and 8% w/w. This group of assays was performed at 50 °C, 20 MPa and constant CO2/EtOH flow rate of 0.50 Â± 0.05 kg/h. Temperature and pressure were chosen considering the extraction yield and AA results from group (1) assays. After the extraction, the EtOH was removed in a rotary evaporator at 40 °C (Fisatom model 802, SÃ£o Paulo-SP, Brazil).
2.4 Soxhlet extraction (SE)
SE with EtOH and with n-hexane were performed. Samples of papaya seeds (10 g) were placed inside a thimble made by thick filter paper and loaded into the 250 mL Soxhlet extractor. The solvent (150 mL) was used at the boiling temperature for extraction (8 h). After extraction, the organic solvents were removed in a rotary evaporator and the extraction yields were calculated.
2.5 Protective effect against lipid oxidation in the vegetable edible oil
The protective effect against lipid oxidation (or antioxidant activity "AA") of SFE and SE extracts was evaluated in EO and compared to AA of the synthetic antioxidant butylated hydroxytoluene (BHT).
2.5.1 EO oxidation.
EO oxidation and measurement of their oxidation products was performed exactly according to our previous work : two edible oil samples without antioxidants (20 g) were placed into an amber flask, and an ethanolic solution of ferrous chloride (Â 10 ÂµL) was added to obtain a concentration of 3.5 mg of Fe2+ per kg of EO. Then extracts of papaya seeds or BHT were added to individual EO samples to give a final concentration of 300 mg of seed extract or BHT per kg of EO , and later the mixture was shaken with Vortex (AP 56 Phoenix, Araraquara-SP, Brazil). The control sample (EO without antioxidants) was prepared the same way and analyzed from the beginning (day zero). The EO samples added with extracts, the control sample and the EO added with BHT were carried to oxidation by heating to 60 Â± 2 °C in an oven for 15 days, every 12 hours the samples were stirring and bubbling for 1 min with oxygen. The lipid oxidation products, such as linoleic acid hydroperoxide (LHP), hexanal (HEX), nonanal (NON) and thiobarbituric acid reactive species (TBARS), were determined quantitatively. The results were presented as the difference of LHP, HEX, NON, and TBARS content between the days zero and fifteen, respectively. Six replicates of the EO oxidation experiment were analyzed, for every essay.
2.5.2 LHP determination
LHP was determined by the conjugated dienes method. Again as in our previous work . Oxidized EO samples were diluted in isooctane and the absorbance was measured at 234 nm (VARIAN Cary 50 Conc, Palo Alto-CA, USA). The LHP concentrations were calculated using its molar extinction coefficient (?=26000 M-1 cm-1) , the result was expressed as mmol of LHP per kg of EO (mmol LHP/kg).
2.5.3 HEX and NON determination
The formation of HEX and NON was quantified by headspace-solid phase microextraction - gas chromatography (HS-SPME-GC).
The samples of EO were put into vials of 20 mL and then these were capped and stirred. The samples were equilibrated at 50 Â± 1 °C with continuos stirring for 20 min. Later, a divinilbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 Âµm) SPME-fiber was exposed to the headspace compounds and these were adsorbed for 30 min. Finally, the compounds were analyzed by gas chromatography with flame ionization detection (GC-FID).
184.108.40.206 GC-FID analysis
The GC system used was again an Agilent Technologies 6820 (Palo Alto-CA, USA), with a 5% phenylpolysiloxane capillary column (30 m, 0.25 mm i.d., 0.1 Âµm film thickness, Agilent J&W Scientific, Folsom-CA, USA). As in our previous work , "The oven temperature was programmed starting at 30 °C for 2 min, increasing from 30 to 60 °C at 2 °C/min and from 60 to 280 °C at 20 °C/min, holding for 2 min. Helium gas was used as carrier gas at a flow rate of 1.8 mL/min. The temperatures of the injector and the detector were 250 and 300 °C, respectively". Splitless injection mode was used for 2 min in the compound desorption from the SPME fiber. Retention times and comparison to standards helped to identify HEX and NON, and the results were expressed as arbitrary area units per mg of EO (AU/mg).
2.5.4 TBARS determination
The TBARS were determined based on Botsoglou method . The results were expressed as mg of malondialdehyde per kg of EO (mg MDA/kg) calculated with a calibration curve of MDA (36 nM to 185 nM).
2.6 Analysis of phenolic compounds by HPLC-DAD
The extracts with highest protective effect against the lipid oxidation of EO were further analyzed by HPLC with DAD detection (Agilent Technologies, Palo Alto-CA, USA, model 1200 with diode array detector). Separation was on a 100 mm x 4.6 mm x 2.6 Âµm KINETEX C18 reverse phase column (Phenomenex) at 35 °C. The mobile phase used was acetic acid 0.3% (A) and acetonitrile (B) at a constant flow rate of 1 mL/min. The eluent gradient used was: 95.5% of A and 4.5% of B during 13 min, then the B concentration was increased until 15% in one min, keeping it for three min, after the B concentration was increased at 22% at a constant rate of 2.3%/min, keeping it for eight min, finally the B concentration was brought up to 100% in two min. Sample injection was 10 ÂµL of a 5000 ppm final extract solution. Some phenolic compounds could be identified by comparison of their UV spectrum and peak retention time to those of pure standard compounds (phenolic acids, catechins, xanthines, and flavonoids).
2.7 Statistic analysis
As mentioned before, six replicates of all experiments (SFE, SE and AA) were performed. The results were reported as mean and standard deviation. In the SFE assay group (1) a 32 factorial design was used and a two-way ANOVA at 95% confidence was carried out in order to find out significant differences between the treatments and the effect of process variables. All analyses were performed using the software R (Version 2.13.0).
The extraction yields obtained by SFE (with pure CO2 and with CO2/EtOH) and SE (with hexane and EtOH) are presented in table 1, together with the CO2 density at the extraction condition used (calculated according to Angus et al. ). The highest extraction yield was obtained in the SE with EtOH (31.46 Â± 0.10%), this yield was higher than that obtained with hexane (22.16 Â± 0.16%) and reported in literature for the SE with petroleum ether (29.16 Â± 0.88%) .
In SFE with CO2 the highest extraction yield was obtained at 60 °C/30 MPa and 50 °C/20 MPa with CO2 densities of 0.8302 and 0.7857 g/mL respectively. The extraction yields obtained at 40 °C/20 MPa, 60 °C/20 MPa and 50 °C/30 MPa were close to these values. Yields obtained at 10 MPa all show very low values, due to the low CO2 density.
Since antioxidant compounds in the papaya seeds might contain compounds with polar groups, additionally, extraction with EtOH modified supercritical CO2 was also performed, at 50 °C/20 MPa. The results obtained (table 1) show that the extraction yield increased with EtOH addition (2, 5 and 8%) as expected, although 2% EtOH addition did not mark a significant yield increase over pure CO2. The extraction yield obtained using CO2/EtOH shows a maximum at 5% EtOH as modifier, which is a behavior similar to studies like reported for Cordia verbenacea leaves  and tamarillo (Solanum betaceum Sendtn) epicarp .
In comparison to the SE with n-hexane the yield values obtained by SFE with pure CO2 were lower, those with CO2/EtOH (5 and 8% EtOH) were higher. Moreover, the yields obtained in this study for Colombian papaya seeds were higher than those reported for the SFE of Brazilian papaya seeds (2.5% at 80 °C/20 MPa) . It might be noted, that the yields reported in this work are all higher than respective values reported for other tropical fruits, like those reported for guava seeds (17.06 Â± 0.05%, at 40 °C/30 MPa, with 10% EtOH) , grape seeds (11.5 % at 40 °C/20 MPa) , and pomegranate seeds (14% at 60 °C/40 MPa) .
The relatively high extraction yields in SFE with CO2, it which are free of solvents, or at maximum with little EtOH contamination (EtOH is generally recognized as a safe solvent) is a good starting point for further evaluation of these papaya seed extracts towards their AA. Hence, in the next section we evaluate the protective effect of papaya seed extracts against lipid oxidation of vegetable edible oil.
3.2 Protective effect against lipid oxidation of vegetable edible oil
The procedure to study the AA of papaya seed extracts is described in section 2.5.1, the resulting AA values are compiled in Table 2. Pure CO2 extracts show in general lowest values of oxidation products for the 20 MPa extractions (10 and 30 MPa a little less effective), with astonishingly little temperature dependence. If compared to the CONTROL extracts, LHP formation is reduced efficiently to low 16%, HEX not very efficiently to 80%, NON and TBARS to the order of 22%. If compared to the synthetic antioxidant, the LHP and HEX results are similar to the results obtained for the synthetic antioxidant BHT but better than the synthetic BHT in the case of TBARS. BHT in the case of NON is clearly less effective than the papaya seed extracts. It is also interesting that Soxhlet extraction with EtOH or with n-hexane is equal or less AA efficient than the seed extracts with pure CO2, less efficient in particular with the LHP formation. Also extracts from SFE with some EtOH, as a more polar modifier, is less efficient (with the exception of TBARS with CO2 at 5% EtOH) than those from SFE with pure CO2 at 20 MPa.
Recently, we isolate and identify the benzylglucosinolate from papaya seed, this compound showed a higher protective effect against lipid oxidation of EO compared to the SE and SFE extracts from papaya seeds . The extracts obtained from papaya seed by SFE with CO2 at 20 MPa showed a greater AA than that reported in the literature for phenolic extracts of guava seed (300 ppm in canola oil) , methanolic extracts from garlic (1000 ppm in sunflower oil) , and methanolic extracts from olive leaf (400 ppm in olve oil) .
The extracts obtained by SE with EtOH and by SFE with CO2 at 50 °C/20 MPa were submitted to HPLC-DAD analysis. The results obtained are showed in the table 3, three phenolic acids and two flavonols were identified in the SE, meanwhile in the SFE were identified five phenolic acids. Caffeic and coumaric acids and kaempferol have been reported in papaya seed extracts , while quercetin, kaempferol, and some phenolic acids (ferulic, caffeic, p-coumaric and p-hydroxybenzoic) have been reported in papaya mesocarp [37,38], also phenolic acids (ferulic, caffeic and p-coumaric) have been identified in papaya skin .
Papaya seeds are an agroindustrial waste of food industry and are not suspect to be human toxic, our goal was to add some value to this waste. This work had shown that is possible obtain a natural extract by green extraction process (using supercritical CO2 at moderate conditions). The supercritical fluid extracts with pure CO2 or CO2/EtOH (e.g. 50 °C and 20 MPa) showed high extraction yields (about 20% w/w) and good protective effect against lipid oxidation of edible oils. These extracts can replace synthetic antioxidants like BHT, in food preservation.
The authors are grateful to the Dirección Investigación Bogotá (DIB) at the Universidad Nacional de Colombia (Project: 201010021085), to Alimentos SAS S.A. (Bogotá-Colombia), to Duquesa S.A. (Bogotá-Colombia) and to the CNPq.
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Table 1. Extraction yields of papaya seed extracts obtained by supercritical fluid and by Soxhlet extraction.
Table 2. Protection against lipid oxidation in vegetable edible oil of papaya seed extracts, as determined from different extraction methods.
Table 3. Phenolic compounds identified in papaya seed extracts obtained by SFE at 50 °C/20 MPa, and SE with EtOH.
Henry I. Castro-Vargasa, Luz P. Restrepo-Sáncheza, Sandra R. S. Ferreirab, Wolfram Baumannc, Fabián Parada-Alfonsoa,*
a Chemistry Department, Universidad Nacional de Colombia, Carrera 30 No 45-03, Bogotá D.C., Colombia.
b Chemical and Food Engineering Department, Universidade Federal de Santa Catarina, EQA/UFSC, CEP 88040-900, Florianópolis, Santa Catarina, Brazil.
c Chemistry Department, Universidad de los Andes, Carrera 1 No 18a-12, Bogotá D.C., Colombia.