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ARTICLE IN PRESSG ModelUPFLU-2978; No. of Pages 5

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Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

j our na l ho me page: www.elsev ier .com/ locate /supf lu

xtraction of bioactive compounds from peach palm pulpBactris gasipaes) using supercritical CO2

aber A. Espinosa-Pardoa, Julian Martinezb, Hugo A. Martinez-Correac,∗

Universidad Nacional de Colombia, Departamento de Ingeniería, Cr 32 #12-00, Palmira, ColombiaFood Engineering Department, Faculty of Food Engineering, University of Campinas, P.O. Box 6121, 13083-862 Campinas, São Paulo, BrazilUniversidad Nacional de Colombia, Cr 32 # 12-00, Palmira, Colombia

r t i c l e i n f o

rticle history:eceived 26 September 2013eceived in revised form 2 March 2014ccepted 7 May 2014vailable online xxx

eywords:

a b s t r a c t

Natural compounds with biological activity have recently attracted special interest in the agro-industryas sources of additives in nutraceutical food production and pharmaceutical industries. Herein, we evalu-ated extracts obtained from peach palm fruit (Bactris gasipaes) using supercritical carbon dioxide, in termsof yield, total phenolic content, total flavonoids, total carotenoids, and antioxidant activity by �-carotenebleaching method. Extractions were performed at 40, 50, and 60 ◦C and 100, 200, and 300 bar; addition-ally, Soxhlet (with petroleum ether) and methanol extraction were conducted. The results showed that

actris gasipaesupercritical fluidiocompoundsarotenoidsntioxidant activity

supercritical CO2 allows obtaining extracts rich in carotenoids and, although it presents lower yield thanconventional extraction (SOX), supercritical CO2 represents a technique with greater advantages. Thebest operation condition for supercritical extraction was 300 bar–40 ◦C, given that the highest concen-tration of carotenoids was obtained, without the yield being significantly different from that obtainedwith 300 bar–60 ◦C, this extract had antioxidant activity comparable to that of commercial caffeic acid.

. Introduction

Bioactive compounds are naturally widespread in the plantingdom because they are synthesized as secondary metabolitesith defense functions, besides being responsible for properties

f color, astringency, and flavor of fruits and vegetables. Theyre increasingly important because given their chemical structure,hese compounds are suitable for scavenging free radicals foundn the human body; said free radicals behave as reactive oxygenpecies (ROS) enabling development of chronic multifactorial dis-ases [1].

These compounds are credited for the mass consumption ofruits due to their high content of antioxidants; with benefi-ial effects in preventing cardiovascular and circulatory diseases,ancer, and neurological diseases, given their anti-inflammatory,nti-allergic, antimicrobial, antithrombotic, and antineoplasticctivity [2,3]. Tropical countries like Colombia and Brazil, becausef their abundance of exotic fruits, have a huge potential for

Please cite this article in press as: F.A. Espinosa-Pardo, et al., Extractionusing supercritical CO2, J. Supercrit. Fluids (2014), http://dx.doi.org/10

he exploitation of the resource to obtain bioactive compoundsf underutilized fruits like the peach palm fruit (Bactris gasi-aes), which could be used as active ingredients in pharmaceutical

∗ Corresponding author. Tel.: +57 2 286 8888; ext. 34350, fax: +57 2 286 8808.E-mail addresses: hamartinezco@unal.edu.co, hamartinez@yahoo.com

H.A. Martinez-Correa).

ttp://dx.doi.org/10.1016/j.supflu.2014.05.010896-8446/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

products, in controlling oxidation processes in food processing, innutraceutical food production with high added value, as well as inthe cosmetic industry [4].

Peach palm fruit (B. gasipaes) is a palm of the Arecaceae family,cultivated in tropical America from Costa Rica to Brazil and Boliviain wet and low zones. It is commonly known as “cachipay”, “chon-taduro”, “pejibá”, and “pununa” (Amazon). The fruit has a fibrousand fleshy mesocarp of deep yellow or orange color; it may be con-sidered a fruit with high nutritional value due to its high contentof fiber, oils, �-carotene, for eight essential amino acids [4–6], andfor its energy value. However, the main feature of the recent inter-est in working with this fruit is the �-carotene content that couldbe obtained from it as a vitamin A precursor of high antioxidantactivity because it can capture free radicals due to its conjugateddouble-bond system [2].

Extraction with supercritical fluids is a technique that uses theproperties of fluids over their critical points to selectively extractsoluble components from raw plant materials; additionally, super-critical carbon dioxide is recognized as an ideal solvent to extractbioactive compounds because it is nontoxic, non-explosive, read-ily available, easy to remove from the final extract, does not causemajor disruptions in biocompounds, and its biological properties

of bioactive compounds from peach palm pulp (Bactris gasipaes).1016/j.supflu.2014.05.010

can be preserved [7–9].This work sought to evaluate bioactive compounds of extracts

obtained from the peach palm fruit using supercritical carbondioxide (Sc-CO2) at different pressures and temperatures. For this

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2 F.A. Espinosa-Pardo et al. / J. of Supercritical Fluids xxx (2014) xxx–xxx

Fcl

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Table 1Extraction conditions.

Experiment Temperature (◦C) Pressure (bar)

E1 40 100E2 40 200E3 40 300E4 50 100E5 50 200E6 50 300E7 60 100

ig. 1. Extraction flowchart [11]. B1: CO2 pump, R1: CO2 reservoir, CA: adsorptionolumn, T: thermocouples, FR: collection bottle, TF: flow totalizer, FI: filter, V1:ocking valve, LF: extraction bed, V2: retention valve, M: manometer, V3: valve.

urpose, the following variables were evaluated: extraction yield,otal phenolic content (TPC), total flavonoids (TF), total carotenoidsnd antioxidant activity (AA%) by �-carotene bleaching (BCB)ethod of each of the extracts obtained.

. Materials and methods

.1. Raw material and chemical characterization

Peach palm fruit grown in el Tambo, Cauca (Colombia) werecquired in the municipal market in Cali (Colombia), then the exo-arp was removed and the mesocarp was cut, lyophilized, ground,acuum packed, and refrigerated until its later use.

Chemical characterization of peach palm fruit (proximate analy-is) for moisture, ash, crude fat, crude fiber, and protein content waserformed by using methods 966.02, 923.03, 920.39, 920.87, and62.09, respectively, from the Official Analytical Chemists Associ-tion (AOAC) [10].

.2. Supercritical extraction

Supercritical extractions were carried out in a supercritical unitFig. 1), which operates up to a maximum pressure of 35 MPa. Thenit has a high-pressure pump for the solvent (Thermo Separationroducts, model 2000, Florida, USA), two programmable thermo-tatic baths (Marconi, model MA-159 and Marconi, model MA-184,iracicaba, SP, Brazil), a flow totalizer (LAO, model G 0.6 ± 0.001 m3,ão Paulo, SP, Brazil), thermocouples, and three control manome-ers (Record (50.0 ± 0.5) MPa, São Paulo, SP, Brazil). The extractioned has an internal 3.41 cm diameter and 46 cm height [11].

All extractions were performed using 10.0 ± 0.010 g of rawaterial, constant CO2 flow of 3.00 l/min for 91 min and a

olvent/raw material S/F ratio (w/w) of 46. The supercritical extrac-ions were performed at different temperature and pressure values;0, 50, and 60 ◦C and 100, 200 and 300 bar, respectively (Table 1).fter extraction, the collection bottles, kept at 5 ± 1 ◦C duringxtraction, were freed of residual CO2, hermetically re-sealed, iso-ated from direct light, and stored under refrigeration (Metalfrioreezer) until its subsequent analysis.

Please cite this article in press as: F.A. Espinosa-Pardo, et al., Extractionusing supercritical CO2, J. Supercrit. Fluids (2014), http://dx.doi.org/10

Additionally, Soxhlet extraction (SOX) was conducted withetroleum ether and methanolic extraction (MET) to comparec-CO2 extraction to traditional methods. For MET, 2 g of peachalm fruit were taken and added to a beaker containing 30 mL of

E8 60 200E9 60 300

methanol (CHEMCO, absolute grade); the solution was stirred for24 h at 25 ◦C; then, the solution was vacuum filtered (0.45 �m fil-ter, Vaccuo Tecnalpump TE-0581) and rotor-evaporated (Heidolph220V). Soxhlet extraction was performed for 6 h using 2 g of rawmaterial and 60 mL of petroleum ether (ECIBRA) as solvent; theether-extract solution was rotor-evaporated (Heidolph 220V) at40 ◦C.

2.3. Extraction yield

The amount of extract obtained (E) in relation to the amountof raw material (RM) used in each type of extraction is a signif-icant factor in assessing bioactive extracts and on the extractiontechniques.

2.4. Chemical characterization: TPC, TF, and total carotenoids

For all Sc-CO2, MET, and SOX extracts total phenols content(TPC) were quantified, expressed as (mg GAE)/g extract throughthe Folin–Ciocalteu method [12]. Total flavonoids (TF) were quanti-fied via spectrophotometric method, according to the methodologydescribed by Zhishen et al. [13]. Gallic acid (Sigma Aldrich) wasused to construct the calibration curve for TPC at different concen-trations and, finally, the following linear equation came about: AbsB = 0.090 + 0.002, R2 = 0.998, where Abs is absorbance (nm) and B isphenolic content (mg mL−1).

Catechin was used as pattern for TF (Sigma Aldrich) and theequation obtained was: C = 0.217 Abs, R2 = 0.999, where C is theflavonoid content (GAE mg mL−1).

Total carotenoid content was determined according to themethodology described by Szydłowska-Czerniak et al. [14] mod-ified. Extract samples (5.0–8.0 mg) were diluted in 10 mL ofn-hexane (96% purity, EMSURE Merck); subsequently, the solu-tion was loaded onto the spectrophotometer (FEMTO 800 XI) at450 nm absorbance, using a 1 cm quartz cell. The calibration curvewas prepared by using standard pattern �-carotene (97.0% purity,Fluka Analytical) at different concentrations ranging from 0.02 to6.1 mg mL−1. The resulting calibration curve was D = 0.006 Abs,R2 = 0.995, where D is the total carotenoid content expressed as�-carotene equivalent (mg mL−1).

�-Carotene bleaching is based on a spectrophotometric methodmonitoring oxidation products due to degradation of linoleic acid.The methodology used was described by Martinez-Correa et al.[15]. Briefly, 5 mL of a dry emulsion of �-carotene and linoleicacid transferred to a test tube and 0.2 mL of extract diluted inethanol, at a 200-�g/mL concentration was added. Similar stan-dard solutions, quercetin and caffeic acid solutions (200 �g/mL)were used as positive controls (standard solutions). The controlsolution was prepared the same way, except that the solution was

of bioactive compounds from peach palm pulp (Bactris gasipaes).1016/j.supflu.2014.05.010

replaced by 0.2 mL of pure ethanolic extract. Both tubes with theextract and the control were subjected to thermal auto-oxidationat 50 ◦C for 120 min and absorbance was measured at 464 nm(spectrophotometer FEMTO 800 XI) at 30-min intervals, against a

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Table 2Composition of peach palm pulp (Bactris gasipaes).

Component Content (%)

Dry matter 30.84Ash 2.64Protein 8.17Oil 18.73

t�cr

A

wa

2

wow

3

3

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bs

Fig. 2. Extraction yield and concentration of carotenoid for experiments. Lower-case letters correspond to significant differences between the different extractionconditions.

0

1

2

3

4

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6

80 10 0 12 0 14 0 16 0 18 0 20 0 22 0 24 0 26 0 28 0 30 0 32 0

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Pressure (bar)

40°C 50°C 60°C

Neutral fiber 8.18Carbohydrates 62.28

arget solution prepared with 5 mL of the emulsion without the-carotene plus 0.2 mL of ethanol. Antioxidant activity (AA%) wasalculated as percentage of inhibition related to the control thatepresents 100% oxidation, using Eq. (1).

A (%) =[

(AC0 − AC

t ) − (Am0 − Am

t )

AC0 − AC

t

]× 100 (1)

here “C” represents the control solution and “m” the sample; A0nd At are the absorbance values at 0 and t minutes.

.5. Statistical analysis

All experiments were performed in duplicate and the resultsere analyzed in the SAS statistical software, managing an array

f completely randomized blocks. A Tukey test was also performedith a confidence range ≥95%.

. Results and discussion

.1. Chemical characterization

As seen in Table 2, which shows the percentage compositionf peach palm pulp, the dry matter content is significantly highompared to other tropical fruits and it is similar to that reportedy Leterme et al. [16] which permits obtaining higher yields ofxtracts; however, a significant variation in the total mineral con-ent was presented from the same study. Compared to Amazonianruit, it was observed that dry matter content is lower [6], but theolombian sample has higher protein content (8.17%) and etherxtract content (18.73%).

Table 2 also shows that the fruit has high lipid content (18.73%),hich together with a reddish color in the mesocarp might indicate

ts high carotenoid content [17,18]. Fiber, carbohydrate, and proteinontents are similar to those reported by Ríos et al. [19] for differentropical Colombian peach palm varieties, showing their potentialor agro-industrialization.

.2. Extraction yield

As seen in Fig. 2, the extraction yield for all Sc-CO2 extractionshowed lower values compared to SOX (up to 394%) and no signif-cant differences with MET (except those at 100 bar). Supercriticalxtraction yields reported by other authors for bioactive com-ounds and/or oils are relatively low compared to those obtained

n this work [8,9,20,21], this may be due to the selectivity andffinity of peach palm soluble compounds in Sc-CO2 compared toonventional extraction methods that obtain higher yields with lit-le selectivity. The disadvantage with MET and SOX extractions ishe limitation in the subsequent recovery, separation, and purifi-ation of the extracts. Additionally, the extract has limitations forood applications due to important reduction of solvents approved

Please cite this article in press as: F.A. Espinosa-Pardo, et al., Extractionusing supercritical CO2, J. Supercrit. Fluids (2014), http://dx.doi.org/10

or this industry [22].The supercritical extraction yield is related to a complex balance

etween the reduction of CO2 density and increased vapor pres-ure of the compounds as temperature increases, which basically

Fig. 3. Global yield isotherms obtained by Sc-CO2 extraction. Experimental pointsconnected for better visualization.

represents the solubility of the bio-compounds in solvent [23].The yield increases with increasing pressure; behavior observedin Fig. 2. However, it is observed that in treatment E9 the patternchanges, introducing the highest yield at 4.9%.

The behavior of the global yield isotherms obtained via SFE at40, 50, and 60 ◦C under different pressures is presented in Fig. 3.As noted, the global yield of extraction decreases with increasingtemperature at 100 bar due to the effect of the decreased density ofthe solvent versus the effect increased vapor pressure of the solute[24,25]. The crossover phenomenon was not observed between theisotherms at 40 and 50 ◦C; however, at 50 and 60 ◦C the crossoverphenomenon occurs at 200 bar, while considering the extractionsperformed at 40 and 60 ◦C the retrograde point is higher, close to250 bar. These values are close to those reported by Filho et al. [21]who also worked on carotenoid extraction.

The yield increases with pressure at three temperatures, thelowest value was 0.19% and was obtained at the lower pressureand higher temperature; an increase of 24.7% was obtained for300 bar–60 ◦C. For 40 and 50 ◦C isotherms yield increases in pres-sure from 200 to 300 bar do not have great influence on extraction,while for 60 ◦C increases in pressure had a significant influence onglobal extraction yield. The statistical analysis performed for onlythe Sc-CO extractions showed significant differences only with

of bioactive compounds from peach palm pulp (Bactris gasipaes).1016/j.supflu.2014.05.010

2the variation of pressure but not with temperature variation, asreported in other studies [9,24,26].

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ARTICLEUPFLU-2978; No. of Pages 5

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.3. Chemical characterization of extracts

Total carotenoids: Carotenoids are the most interesting bioactiverganic pigments for fruits like peach palm, which have characteris-ic reddish color and are rich in oil; these pigments are responsibleor the color of the fruit are vitamin A precursors, while collab-rating in immune system regulation, oxidation inhibitors, anderoxidation of fats [27].

Fig. 2 shows that for supercritical extraction carotenoid concen-ration in the extracts increases as the yield rises; it means that its increased with pressure and decreases with increasing temper-ture. The E9 treatment (300 bar–60 ◦C) allows obtaining a higherxtraction yield and significant carotenoid content. The carotenoidontent in the extracts obtained from treatments E1–E9 reachedaximum values at highest pressure, and decreased with increas-

ng temperature, which may be due to carotenoid sensitivity tohermal degradation [18]. This same behavior was also reported byracia et al. [26].

Regarding conventional extractions, SOX showed the highestoncentration of all extracts (4.157 mg/g), with significant differ-nces compared to MET and Sc-CO2. Because of its non-polarature, it presents affinity for carotenoids and allows for higherields, as reported by Cadoni et al. [27] and Vági et al. [28]. Thepposite occurs with MET, which is a polar solvent and gets theowest concentration of carotenoids. For both cases, the extractsre not entirely free of residual solvent, and the use of petroleumther as solvent is not recommended for some applications becausef its carcinogenicity.

The ANOVA for carotenoid content showed that both tempera-ure variation and pressure produce significant differences in thenal carotenoid concentration in the Sc-CO2 extracts; however, thexperiments conducted at 300 bar and 40 and 50 ◦C showed no sig-ificant difference from those obtained at 200 bar–40 and 50 ◦C;ence, for extractions aiming to recover carotenoids, it is enougho work at 200 bar and 40 ◦C to obtain the high carotenoid content2.01 mg/gE).

Total phenolic and total flavonoid tests were not detected viapectrophotometry (TPC, TF <0.0102 mg mL−1) in the extracts. Con-reras et al. [29] reports total phenol content for peach palm fruitignificantly lower compared to other Colombian exotic fruits, with

value of 65.7 mg GAE/100 g of fresh raw material; additionally,he non-polar nature of Sc-CO2 solubilizes and extracts basicallyon-polar compounds, which correspond to the lipid fraction ofhe array. It is not an effective method to obtain extracts rich inhenolic compounds with polar nature [30].

The use of polar solvents like methanol allows the extractionf phenolic compounds of polar nature. Martinez-Correa et al.15] reported that primary extraction of the lipid phase of plant

atrix (for example Sc-CO2) facilitates subsequent removal of theolar compounds (e.g., phenolic) using conventional solvents likethanol and water, thereby, obtaining more concentrated polyphe-ol extracts.

.4. Antioxidant activity (AA)

Fig. 4 shows the antioxidant activity (AA%) obtained by MET, SOXnd Sc-CO2 extracts at 60 min; additionally, compared to standardntioxidants (caffeic acid and quercetin). For supercritical extracts,he highest AA (%) value was obtained by E9 (10.3%) and its activitys statistically similar to that presented by the standard caffeic acid.

ET also presented a statistically equal value, although it presentedhe lowest concentration of carotenoids of all types of extracts.

Please cite this article in press as: F.A. Espinosa-Pardo, et al., Extractionusing supercritical CO2, J. Supercrit. Fluids (2014), http://dx.doi.org/10

The highest antioxidant activity was obtained by the SOXxtract, reaching 25% inhibition; it also presented the highestverall extraction yield and the highest carotenoids concentra-ion, which could be related to the high antioxidant activity. The

Fig. 4. Antioxidant activity of the different extracts. Lowercase letters correspondto significant differences between the different extraction conditions.

non-polar nature of the compounds in SOX extract allows thatduring the test, these can be in the water/lipid interface, thus, pro-tecting the emulsion and inhibiting oxidation of �-carotene [15].The same situation is explained for the AA value obtained for stan-dard quercetin, which scored 26%.

Extracts resulting from supercritical technology presentedlower ability to inhibit oxidation reactions than the MET extract,even with a greater carotenoid concentration than MET. This behav-ior suggests that the antioxidant activity determined by the DBCmethod would not be related to the carotenoid content in theextracts, as reported by Rodriguez-Amaya [31].

4. Conclusions

This study allowed showing that the use of supercritical CO2 pro-duces carotenoid-rich extracts from peach palm pulp and, althoughpresenting lower yield than conventional extraction (SOX), theSc-CO2 technique offers advantages compared to conventionalextraction. The recommended operating condition for supercriticalextraction is 300 bar–40 ◦C because it allows obtaining the high-est carotenoid concentration. The Sc-CO2 extracts obtained an AA(%) comparable to standard antioxidants. The importance of �-carotene is mainly as vitamin A precursor, which provides severalbenefits to the human organism operation; as a natural colorantin the food industry, making this bio-compound an alternative forfood (nutraceuticals) and pharmaceutical products.

Acknowledgements

The authors thank the staff at the LASEFI Laboratory in Brazil fortheir cooperation in this research.

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