antioxidant properties of aqueous and ethanolic extracts of tara ( caesalpinia spinosa ) pods in...

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Research ArticleReceived: 29 May 2013 Revised: 22 July 2013 Accepted article published: 8 August 2013 Published online in Wiley Online Library: 13 September 2013

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6335

Antioxidant properties of aqueous andethanolic extracts of tara (Caesalpinia spinosa)pods in vitro and in model food emulsionsMonika Skowyra,a Vıctor Falguera,b Gabriela Gallego,a Sara Peiroa

and Marıa Pilar Almajanoa∗

Abstract

BACKGROUND: The successful replacement of some synthetic food antioxidants by safe natural antioxidants has fosteredintensive search for new vegetable sources of antioxidants. In our study the phenol and flavonoid content of extracts oftara pods was determined. The antioxidant activity was also studied by three different analytical assays: the measurement ofscavenging capacity against a radical ABTS+, the oxygen radical absorbance capacity (ORAC) and the ferric reducing antioxidantpower (FRAP).

RESULTS: All analyzed samples showed a good antioxidant capacity, but the use of a solution of ethanol 75% in a 1 h ultrasonicprocess allowed achieving the greatest quantity of phenolics (0.464 mg gallic acid equivalent (GAE) g−1 dry weight (DW) ) andthe highest antioxidant activity measured by the ABTS+ and ORAC methods (10.17 and 4.29 mmol L−1 Trolox equivalents (TE)g−1 DW, respectively). The best method for efficient extraction of flavonoids (3.08 mg catechin equivalent (CE) g−1 DW) was a 24h maceration in cold water. Two extracts obtained with ethanol 75% and water were added to a model food system (oil-in-wateremulsion) and the oxidative stability was studied during storage at 38 ◦C. Oxidation was monitored by determination of theperoxide value. The addition of 48 µg mL−1 ethanol extract to the emulsion delayed oxidation to the same extent as 17.8 µgmL−1 of Trolox, while water extract was only effective in the early stages of the oxidation process.

CONCLUSION: The results of this study indicate that ethanolic tara extracts may be suitable for use in food, cosmetic andnutraceutical applications.c© 2013 Society of Chemical Industry

Keywords: Caesalpinia spinosa; phenolics; flavonoids; emulsions; peroxide value

INTRODUCTIONThe tara tree (Caesalpinia spinosa), which belongs to theCaesalpinaceae family, is a native species of Peru that is widelydistributed in Latin America. Tara is a small tree that is 2–3 m inheight with flat, oblong indehiscent orange pods that contain fourto seven rounded seeds.1 Tara seeds are the source of a thickeninggum used in several foods and in paint. In addition, tara podsare ground and used as raw material for the extraction of severalcompounds that have interesting applications in cosmetics,medicine and the chemical and pharmaceutical industries.2

Moreover, its extracts have been reported to have antitumor,

antimicrobial and antioxidant activities.3–5

Tara tree was traditionally considered the second tannin feed-stock after Schinopsis balansae.6 Tannins are common secondarymetabolites in vascular plants, developing functions related toplant defense, from physical damage to pathogen attacks. Onthe basis of their structural characteristics, tannins are classifiedinto two main groups: hydrolysable tannins (HTs) and condensedtannins (CTs). In HTs, a carbohydrate (usually α-glucose) is partiallyor totally esterified with phenolic molecules such as gallic acid(giving gallotannins) or ellagic acid (giving ellagitannins).7 On theother hand, CTs are oligomeric or polymeric flavonoids based onflavan-3-ol units (commonly catechin or epicatechin) linked via a

carbon–carbon bond.8 Although tara pod composition has not yetbeen clearly established, its beneficial properties have been mainlyattributed to the presence of HTs.9 Among them, tannic acid iswell known for its ability to induce beneficial effects on humanhealth through the expression of some biological activities.10

Recent studies have reported other properties of tannins thatmake them suitable for use as an astringent agent, for eliminatingparasites and as an antipyretic.11 In addition, its ability to reduceserum cholesterol and triglycerides, and to suppress insulin-

stimulated lipogenesis, has been documented.12–14 Meanwhile,other studies revealed that its antioxidant activity seems to becorrelated with its copper-chelating capacity.15

In general, it is known that plant extracts containing tannins areable to interact with biological systems through the induction ofsome of the aforementioned effects. Accordingly, some extracts

∗ Correspondence to: Marıa Pilar Almajano, Chemical Engineering Department,Technical University of Catalonia, Av. Diagonal 649, 08034 Barcelona, Spain.E-mail: [email protected]

a Chemical Engineering Department, Technical University of Catalonia, 08034,Barcelona, Spain

b Institut de Recerca i Tecnologia Agroalimentaries (IRTA), 25198, Lleida, Spain

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have recently been distributed commercially in order to takeadvantage of their properties.16,17 In addition, such distributionis expected to grow rapidly in the next years due to a fosteredinterest in searching for new sources of natural antioxidants thatare safe, cheap and without the adverse effects of syntheticones.18,19 These new natural extracts should have the ability toact either directly taken as a diet complement or as an additive infood systems, fulfilling additional technological functions such ascationic dye removal6 or increasing the oxidative stability of oilsand oil-in-water emulsions.20

For all these reasons, tara pods have a high potential for medical,industrial and food applications. Their content of gallic acid andtannins, among other compounds, makes them a suitable rawmaterial for preparing a wide variety of extracts. Thus this work hastwo main aims: (i) to search for the best extraction method fromtara pods to take advantage of their antioxidant and antiradicalproperties; and (ii) to assess the usefulness of these extracts asoil-in-water emulsions antioxidant.

MATERIALS AND METHODSRaw materialFruits pods of Caesalpinia spinosa (tara) were a commercial productfrom Peru (Mabrata Tara Powder, Agrotara SAC). Refined sunfloweroil was purchased in a local market.

Reagents6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox),gallic acid, phosphate-buffered saline (PBS), 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid diammonium salt (ABTS),2,2,-azobis(2-methylpropionamide) dihydrochloride (AAPH), fluo-rescein (C20H10Na2O5) and 2,4,6-tripyridyl-s-triazine (TPTZ) werepurchased from Sigma-Aldrich Co. Ltd (Gillingham, UK). Folin–Ciocalteu reagent, absolute ethanol, aluminium oxide, ferric chlo-ride (FeCl3), ammonium thiocyanate (NH4SCN), anhydrous sodiumcarbonate and Tween 20 were analytical grade from Panreac(Barcelona, Spain). Deionized and pure water (Millipore-Q System)was used for the study.

ExtractionAir-dried and ground to a fine powder, tara pods were weighed (3 g)and extracted with 60 mL of solvent. Water and ethanol–watermixtures at 75:25 and 50:50 (v/v) were selected as extractionsolvents. Two different methods, described below, were usedto check which solvent could extract the maximum amount ofpolyphenolic compounds: (i) the solution was stirred continuouslyfor 24 h at 4 ◦C; (ii) the solution was ultrasound processed in a bathsonicator for 1 h at room temperature.

The extracts, prepared in triplicate, were centrifuged (Sigma6K10, Osterode, Germany). Part of the supernatant was taken forsubsequent use to determine the antiradical capacity. The volumeof the remaining supernatant was measured and the solutionwas evaporated, frozen at −80 ◦C for 24 h and lyophilized for 3days. Samples were then weighed and protected from light in adesiccator until used to prepare an oil–water emulsion system.

Total phenol and flavonoid contentTotal polyphenol content of extracts was determined bycolorimetric spectrophotometry following the Folin–Ciocalteumethod,21 slightly modified and adapted for microplates. Sampleswere taken from the extract solutions, diluted 1:30 (v/v) and

Folin–Ciocalteu reagent (4% by volume), sodium carbonatesolution 20% (30.8% by volume) and Milli-Q water were added.Samples were well mixed and left in the dark for 1 h. Absorbancewas measured at 725 nm using a UV–visible spectrophotometer(Fluostrar Omega, PerkinElmer, Paris, France) and the results wereexpressed in gallic acid equivalents (GAE), using a gallic acidstandard curve (10–70 µmol L−1).

Flavonoid content was determined as described by Bonvehiet al.,22 with some modifications. An appropriate dilution of theextract was mixed with the same volume of 2% AlCl3 in methanolsolution (5% acetic acid in methanol). The mixture was allowed toreact for 10 min and the absorbance was read at 430 nm againsta blank sample without reactants. Values were determined from acalibration curve prepared with catechin (ranging from 6 to 60 mgL−1) and expressed as milligrams of catechin equivalent per gramof dry weight.

Antioxidant capacity determinationABTS assayThe first method used was the ABTS+ (radical cation) discolorationassay.23 The assay is based on the ability of an antioxidantcompound to quench the ABTS+ relative to that of a referenceantioxidant such as Trolox. A stock solution of ABTS+ radical cationwas prepared by mixing ABTS solution with potassium persulfatesolution at 7 and 2.45 mmol L−1 final concentrations, respectively.The mixture was maintained in the dark at room temperature for16 h before use. The working ABTS+ solution was produced bydilution in 10 mmol L−1 PBS (pH 7.4) incubated at 30 ◦C of thestock solution to achieve an absorbance value of 0.7 (±0.02) at734 nm. An aliquot of 20 µL of diluted extract was added to ABTS+working solution (180 µL). For the blank and standard curve, 20µL PBS or Trolox solution was used, respectively. Absorbance wasmeasured by means of a UV–visible spectrophotometer (FluostarOmega, PerkinElmer) at 734 nm immediately after addition andrapid mixing (t0) and then every minute for 15 min. Readings att = 0 min (t0) and t = 5 min (t5) of reaction were used to calculatethe inhibition percentage value for each extract:

% inhibition of the blank = t0 − t5

t0100

% inhibition of the sample = t0 − t5

t0100

− % inhibition of the blank

A standard reference curve was built by plotting percentinhibition value against Trolox concentration (2–32 µmol L−1).The radical-scavenging capacity of extracts was quantified asmillimoles per liter of Trolox equivalent per gram of dry weight.

Oxygen radical absorbance capacity (ORAC) assayThe ORAC method was adapted from Ou et al.24 The assay wasperformed with an automated microplate reader and 96-wellplates. Diluted extract (40 µL) was transferred by pipette intoeach well and then 120 µL of 1.34 µmol L−1 fluorescein workingsolution in phosphate buffer (13.3 mmol L−1) at 37 ◦C were addedto each sample. The plate was placed in a spectrophotometer(Fluostar Omega, PerkinElmer) and incubated at 37 ◦C. The initialfluorescence was recorded at an excitation wavelength of 485nm and an emission wavelength of 535 nm. AAPH, 40 µL,

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30 mmol L−1) was then added to each sample well andthe fluorescence was measured immediately and every 2 minthereafter for 120 min. For the calibration curve, solutions of Troloxwere prepared in the range 8–58 µmol L−1. The ORAC value foreach extract was calculated using a regression equation relatingTrolox concentration and the net area under the fluorescencedecay curve (AUC):

AUC =

0.5 +∑Nc

i=1fi

fi

tc

Decreasefluorescence = AUC − AUCBl

where AUC = area under the curve of the sample in the well;AUCBl = area under the curve of the blank; fi = fluorescence units(f 1 is the value of the first reading); Nc = number of cycles; andtc = time of each cycle, in this case tc = 2 (2 min).

Results are expressed as millimoles per liter of Trolox equivalentsper gram of dry weight.

FRAP assayThe FRAP method was performed as described by Benzie andStrain,25 with some modifications. The FRAP reagent was preparedwith acetate buffer (300 mmol L−1, pH 3.6), TPTZ (10 mmol L−1 inHCl, 40 mmol L−1) and FeCl3 (20 mmol L−1). The proportions were10:1:1 (v/v/v), respectively. A suitable dilution of the extract wasadded to the FRAP reagent (1:30, v/v) and incubated at 37 ◦C. Theassay was performed by means of an automated microplate reader(Fluostar Omega, PerkinElmer) with 96-well plates. The absorbanceat 593 nm at time zero and after 4 min was recorded. The analysiswas performed in triplicate for each triplicate extract and valueswere determined from a calibration curve of Trolox (ranging from2.5 to 33 µmol L−1). The results are expressed as millimoles perliter of Trolox equivalents per gram of dry weight (mmol L−1 Troloxg−1 DW).

Determination of gallic acid by high-performance liquidchromatography (HPLC)HPLC analyses of the tara pod extracts were carried out using anAcquity UPLC System (Waters, Milford, MA, USA) with photodiodearray (PDA) detector. Tara pod extracts (10 µL) were injected on toan analytical C18 column (Symmetry, 5 µm, 3.9 × 150 mm, Waters)and the column temperature was set to 25 ◦C. The mobile phasewas composed of 0.5% formic acid (v/v) in acetonitrile (eluent A)and 0.5% formic acid (v/v) in water (eluent B). The gradient programwas as follows: 5% A (6 min), 5–95% A (2 min), 95% A (5 min), 95–5%A (1 min), 5% A (6 min). Total run time was 20 min. The detection wasmade at 273 nm for gallic acid. The standard was identified by itsretention time and its concentration was calculated by comparingthe peak area of samples with that of the standard. Standardsolutions with concentrations ranging from 10 to 100 ppmwere then prepared by diluting the stock standard solution withwater. The tara pod extracts obtained using different extractionconditions were filtered through a 0.45 µm filter for HPLC analysis.

Oil-in-water emulsion systemAntioxidant evaluation in oil-in-water emulsionOil-in-water emulsions were prepared with 1% Tween 20 asemulsifier and 10% sunflower oil triacylglycerols, previously filteredthrough alumina as described by Yoshida et al.26 in order toremove the tocopherols. The oil was added dropwise to the

aqueous sample containing emulsifier cooled in an ice bath, whilesonicating for 5 min in total. Freeze-dried powder of the extractof tara was redissolved in ethanol 50% (v/v) and added directlyto the emulsion and homogenized, obtaining final concentrationsof 12, 24 and 48 µg mL−1 (C1, C2 and C3, respectively). For thenegative control no extract was added, and the positive controlwas prepared with Trolox (17.8 µg mL−1) dissolved in ethanol.All emulsions were stored in triplicate in 30 mL amber bottlesin the dark and allowed to oxidize at 38 ◦C. Peroxide value (PV)was measured periodically using aliquots of 0.005–0.1 g of eachsample and determined by the ferric thiocyanate method,27 aftercalibrating the procedure with a series of oxidized oil samplesanalyzed by AOCS Official Method Cd 8-53.28

The pH of the samples was measured (pH meter GLP21, CrisonInstruments, Barcelona, Spain) as a parameter to investigate itscorrelation with PV.

Principal component analysis (PCA)PCA was carried out in order to discover the correlation betweenthe analyzed compounds and activities and the used extractionmethods and solvents. This multivariate procedure was performedby means of The Unscrambler v. 10.1 (Camo Process AS, Oslo,Norway).

RESULTS AND DISCUSSIONTotal polyphenol contentTotal polyphenols were quantified in the extracts in order tocompare the three solvents and the two extraction methods(Table 1). The best polyphenol extraction was achieved usingethanol 75%. Meanwhile, multiple range tests showed that nosignificant differences existed between passive (24 h in therefrigerator) and active (1 h ultrasonic) extraction with thissolvent (P > 0.05). Conversely, the lowest polyphenol amountwas extracted using water as solvent, regardless of the method.On average, ethanol 75% allowed an increase of 31.4% in theperformance of the extraction when compared with water. Indeed,PCA (in which the two first principal components explained 74% ofthe overall variance) revealed that phenolic content was stronglyand negatively correlated with water, while the highest positivecorrelation was that with ethanol 75% (Table 2). In addition, thelowest correlations were those relating to the extraction method.

The use of ethanol 75% allowed an average extraction of 464mg GAE g−1 ground dry tara pods. Similarly, Veloz-Garcıa et al.29

reported a polyphenol extract yield of 48 ± 3% of dry weight incascalote (Caesalpinia cacalaco) pods, a plant of the same genus,using a mixture of water–methanol–acetone (8:1:1). Almost 90%of this phenolic extract was found to be gallic acid, while theremaining 10% was tannic acid. Galvez et al.30 found a yield of25% in gallic acid (dry weight) after a hot basic hydrolysis and acidcrystallization. However, Romero et al.1 reported an extractionof only 44 mg GAE g−1 ground tara pods after a process withsupercritical CO2. Consequently, the extraction method and thesolvent used play a key role in the extraction of polyphenolsfrom Caesalpinia sp. pods. Likewise, Seabra et al.31 studied theextraction of phenolics from tara seeds with different pressurizedCO2 –ethanol–water mixtures and stated that the best yields werealso achieved with ethanol-rich solvents (extraction in differentdegrees of ethanol–water mixtures at 50:50 to 98:2 (v/v), whilewater and CO2 had antagonistic effects. Whatever the consideredextraction method or solvent, tara pods were shown to have a

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Table 1. Polyphenol and flavonoid content and antioxidant activity of the different tara pod extracts

Antioxidant activity

(mmol L−1 TE g−1 DW)

Extraction Solvent

Polyphenol content

(g GAE g−1 DW)

Flavonoid content

(mg CE g−1 DW) ABTS ORAC FRAP

24 h refrigerator Water 0.3497a ± 0.0076 3.08f ± 0.02 6.90a ± 0.28 2.29a ± 0.28 4.66b ± 0.06

Ethanol (50%) 0.4453c ± 0.0086 2.89d ± 0.03 7.37b ± 0.03 4.19bc ± 0.24 5.99d ± 0.06

Ethanol (75%) 0.4602d ± 0.0180 2.93e ± 0.02 9.60c ± 0.09 3.98b ± 0.38 3.89a ± 0.29

1 h ultrasonic Water 0.3565a ± 0.0006 2.48c ± 0.04 6.80a ± 0.21 2.55a ± 0.42 4.59b ± 0.05

Ethanol (50%) 0.4316b ± 0.0085 1.30a ± 0.03 6.91a ± 0.02 4.22bc ± 0.17 5.55c ± 0.07

Ethanol (75%) 0.4676d ± 0.0151 1.79b ± 0.02 10.17d ± 0.20 4.29c ± 0.18 3.81a ± 0.12

Results are expressed as mean value ± standard deviation. Different letters within each column indicate significant differences according to multiplerange tests (confidence level 95%).

Table 2. Correlations between the analyzed compounds and activities and the extraction methods and solvents, from principal component analysis

Solvent Method

Flavonoids ABTS ORAC FRAP Water Ethanol 50% Ethanol 75% Ultrasonic Cold maceration

Phenolics −0.3253 0.7392 0.9605 −0.0977 −0.9705 0.2964 0.6741 0.0017 −0.0017

Flavonoids −0.0790 −0.4853 −0.0709 0.3979 −0.3421 −0.0558 −0.8478 0.8478

ABTS 0.5438 −0.7194 −0.5668 −0.4173 0.9842 0.0020 −0.0020

ORAC 0.1329 −0.9893 0.5237 0.4655 0.1213 −0.1213

FRAP −0.1087 0.9034 −0.7947 −0.1258 0.1258

remarkably higher polyphenol content than that reported forcarob (Ceratonia siliqua L.) pods32 and for Acacia polyacantha, A.tortillis, A. nilotica and Dichrostachys sp. pods.33

Total flavonoid contentFlavonoids represent approximately two-thirds of the dietaryphenolics, being also the most important group regarding theirbiological activities.34 Of these, the most abundant moleculesin the diet are flavonols (catechins plus proanthocyanidins),anthocyanins and their oxidation products. As far as flavonoidextraction from tara pods is concerned (Table 1), significantdifferences were found depending on both the extraction methodand the solvent. The highest content was obtained in the extractperformed with water during 24 h in the cold (3.08 mg CE g−1).This method provided in all cases better results for flavonoidextraction than ultrasonication for 1 h, regardless of the solvent.In the same way, pure water provided better results than ethanol,which was expected due to the fact that flavonoids are mainlywater-soluble compounds.35 Both facts can also be observed inthe PCA correlations (Table 2).

Although no flavonoid concentration in tara pods has beenreported to date, it was expected to be high due to their tannincontent, since chemical degradation of tannins leads to flavanolsincluding catechin, among several others.36 In carob (Ceratoniasiliqua L.) pods, a remarkably lower flavonoid content has beenreported, being between 0.41 and 0.48 mg CE g−1 dry weight.32,37

Antioxidant activityThe high phenolic and flavonoid content of tara pods extracts maybe a good indicator of their antioxidant capacity. Antioxidantproperties of phenolic compounds have been thoroughlydescribed, although in some cases the mechanism of such activity is

not yet fully understood. Some of the biological functions leadingto this effect involve phenolic compounds acting as hydrogendonors, free radical acceptors, chain oxidation reaction interruptersor metal chelators.38 On the other hand, antioxidant activityhas been quantified in different extracts from pods of Acaciapennatula,39 Acacia auriculiformis,40 Acacia nilotica,41 Ceratoniasiliqua L.,37 Erythrina lysistemon,42 Arachis hypogaea L.,43 Phaseolusvulgaris L.44 and Caesalpinia cacalaco,29 among others.

Antioxidant activity of the extracts from tara pods was assessedby three different methods: ABTS+, ORAC and FRAP. Using severalmethods provides more comprehensive information about theantioxidant properties of the original product.45 The highest func-tionality measured via ABTS+ discoloration was found in ethanol75% extracts, being slightly higher if the process had been carriedout with ultrasound (10.17 TE mmol L−1 g−1) rather than withcold maceration (9.60 TE mmol L−1 g−1). The activity of the otherextracts, regardless of the solvent and method, was remarkablylower: between 6.90 and 7.37 TE mmol L−1 g−1. ABTS+ quenchingactivity was positively correlated with total phenolic content, aswell as with ethanol 75% (Table 2), confirming the previouslystated conclusion. As far as the ORAC assay is concerned, resultsdepended mainly on the solvent used, although the highestactivity (4.29 TE mmol L−1 g−1) was found again in the sampleextracted with ethanol 75% with ultrasound. However, no signif-icant differences were found between ethanol 50% and ethanol75% in either of the two methods (P > 0.05). Meanwhile, extractsperformed with water provided the lowest ORAC values. TheORAC assay was the antioxidant assessing method best correlatedwith total phenolic content in the extracts (0.9605; Table 2). If alinear (univariate) regression is carried out between the meansof phenolic content and ORAC values, the resulting model is sig-nificant, with a P-value of 0.0023 and a determination coefficient(R2) of 0.9225. Finally, opposite to what has been described for


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Table 3. Effect of different extraction methods on gallic acid contentof tara pod extracts

Extraction Solvent mg GA g−1 DW

24 h refrigerator Water 33.392 ± 1.8

Ethanol (50%) 22.289 ± 0.2

Ethanol (75%) 16.925 ± 0.6

Results are expressed as mean value ± standard deviation.

ABTS+ and ORAC methods, the FRAP assay revealed the lowestferric-reducing ability of the extracts performed with ethanol75% (3.85 TE mmol L−1 g−1 on average). In addition, ethanol50% showed better activity than water, being in turn higher inthe cold maceration extract (5.99 TE mmol L−1 g−1). However,FRAP had the weakest correlations with phenolic and flavonoidcontent.

Previous reported antioxidant assays on extracts from pods ofCaesalpinia sp. are very limited. Romero et al.1 found an activityof 0.83 µg GAE mL−1 of supercritical CO2 tara polyphenol extractassessed via DPPH assay. However, as previously commented, thepolyphenol content in that extract was much lower than in thepresent work, probably due to the extraction method used. Veloz-Garcia et al.29 described the antioxidant (β-carotene–linoleatemethod) and antiradical activity (DPPH) of the phenolics extractedfrom Caesalpinia cacalaco, finding dose-dependent, comparablevalues to those of pomegranate seeds.

Martinez et al.45 assessed the antioxidant activity of Theobromacacao L. by means of the ABTS, DPPH and FRAP methods,obtaining in all cases lower values (of the order of µmol L−1

TE g−1) than those found for tara in this study. Again, also thephenolic content reported for those samples was remarkably lower(between 80.17 and 365.33 mg 100 g−1 depending on the sourceand the solvent). The same authors also found a good univariatecorrelation between total phenolics and the results of both ABTSand DPPH assays. However, a good correlation was also reportedbetween phenolics and FRAP, which has not been found in thiswork. Regarding carob (Ceratonia siliqua L.) pods, a well-known

source of antioxidant phenolics, Makris and Kefalas37 found thatits extracts exhibited higher activity (around 1.3 mmol L−1 TE) thanaged red wine tannins and tannic acid, of the same scale as that ofcatechin and lower than that of gallic acid, quercetin and caffeicacid. However, the results found in tara pods extracts were evenhigher than these reported for carob pods.

Quantitative analysis of gallic acidThe results (Table 3) revealed that the gallic acid concentrationranged from 16.9 to 33.4 mg g−1 DW. Water was found to yieldthe extract with the highest gallic acid content; conversely, whenethanol 75% was used as extraction solvent the content of gallicacid was the lowest. Extracts performed with ethanol providedthe lowest gallic acid content, because the majority of extractedcompounds can be derivatives of this molecule. The galloylquinicacids of tara tannin can be divided into different groups: mono-, di-,tri- and tetragalloylquinic acids that lack depsidic galloyl residues;depsides related to the 3,4,5-trigalloyl structure were previouslyconsidered to be the major components of tara tannin.46

Oil-in-water emulsionsBesides their effects on human health through direct ingestion,one of the main applications of antioxidant extracts is their use inthe food industry to avoid or delay oxidation of perishable systems.In this study, this technological ability has been assessed in oil-in-water emulsions as a model food, testing water and ethanol 75%extracts performed in cold maceration.

Peroxide valueThe formation of hydroperoxides (Fig. 1) was significantly faster inthe sample without any antioxidant, reaching 40.5 meq kg−1 afteronly 4 days (P < 0.05). This value was exceeded at the 14th dayin the control with 17.8 µg mL−1 Trolox, while the samples withtara extracts offered prevention from oxidation between these. Asexpected from the results discussed in the previous section, theextracts carried out with ethanol 75% provided better protectionthan water extracts at the same concentration. As an example,after 1 week peroxide value was 122.5 meq kg−1 in the negative

0

20

40

60

80

100

120

140

160

180

0 5 10 15 20

PV

(m

eq k

g-1)

Storage time (days)

Control Tara_Et_C1 Tara_Et_C2 Tara_Et_C3

Tara_W_C1 Tara_W_C2 Tara_W_C3 Trolox

Figure 1. Evolution of primary oxidation (peroxide value) in model food system (oil-in-water emulsion, 10% oil) with different concentrations of taraextracts. Et, extract performed with ethanol 75%; W, extract performed with water. C1, 12 mg mL−1; C2, 24 mg mL−1; C3, 48 mg mL−1.


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0 100 200 300 400

20

40

60

80

Time (h)

PV

(m

eq k

g-1

)

Trolox

Tara_W_C3

Tara_W_C2

Tara_W_C1

Tara_Et_C3

Tara_Et_C2

Tara_Et_C1

CTR

Figure 2. Time to reach different peroxide values (PV) in model food system (oil-in-water emulsion 10% oil) with different concentrations of tara extracts.Et, extract performed with ethanol 75%; W, extract performed with water; C1, 12 mg mL−1; C2, 24 mg mL−1; C3, 48 mg mL−1.

0

1

2

3

4

5

6

7

8

0 5 10 15 20

pH

Storage time (days)

Control

Tara_Et_C1

Tara_Et_C2

Tara_Et_C3

Tara_W_C1

Tara_W_C2

Tara_W_C3

Trolox

Figure 3. Evolution of pH in model food system (oil-in-water emulsion, 10% oil) with different concentrations of tara extracts. Et, extract performed withethanol 75%; W, extract performed with water; C1, 12 mg mL−1; C2, 24 mg mL−1; C3, 48 mg mL−1.

control, 100.7 meq kg−1 in 12 µg mL−1 water extract (W_C1), 49.3meq kg−1 in 12 µg mL−1 ethanol 75% extract (Et_C1), 18.8 meqkg−1 in 48 µg mL−1 water extract (W_C3), 2.2 meq kg−1 in 48 µgmL−1 ethanol 75% extract (Et_C3) and 1.9 meq kg−1 in the positivecontrol. The most remarkable issue is that, as observed in Fig. 1,the behavior of the emulsion with 48 µg mL−1 of ethanol 75%extract was the same as the positive control with Trolox until theend of the experiment (after 18 days).

If the induction time is defined as the time in which the sampleremains under 20 meq hydroperoxides kg−1, this parameter takes12 days for the Trolox control and the Et_C3 sample, 8 days forEt_C2, 5 days for Et_C1, 4 days for W_C2 and W_C1 and 1 day for thenegative control. When the time to reach higher PV is calculated(Fig. 2), the same order is maintained. For all values, 48 µg mL−1

of the extract of tara pods performed with ethanol 75% led tothe same delay in oxidation as 17.8 µg mL−1 of Trolox. Kiokiaset al.20 reported peroxide values between 45.60 and 51.15 meqkg−1 after 2 months in 10% sunflower oil-in-water emulsions with2 g L−1 of different carotenoids including β-carotene, lycopene,paprika, lutein and bixin. Although the time to reach these valueswas remarkably shorter in this study, also the concentration ofantioxidant extracts was much lower (between 12 and 48 mg L−1).Maisuthisakul et al.47 reported that a 10% oil-in-water emulsionwith 100 mg kg−1 teaw (Cratoxylum formosum Dyer) extract took

4.55 days at 60 ◦C to reach a PV of 50 meq kg−1. In this work, theemulsion with 48 mg L−1 of the ethanol 75% extract exceededthis value at the 14th day at 38 ◦C. Ramful et al.48 found thatEugenia pollicina leaf extract at a concentration of 0.02% was alsoeffective in slowing down hydroperoxide formation in soybeanoil emulsion during 13 days of storage at 40 ◦C. Roedig-Penmanand Gordon49 reported that tea extracts added to sunflower oil-in-water emulsion were very effective in their stabilization, the teaextract (0.03%) being similar to BHT (0.02%) and taking 40 days ofstorage at 30 ◦C to reach a PV of 30 meq kg−1.

pHHydroperoxides are the main primary products of lipid oxidation,but they are highly unstable and easily break up into secondarycompounds, resulting in the appearance of aldehydes, ketones,epoxides or organic acids, which may lead to changes in the pH.50

In addition, since it is known that many antioxidant molecules areless effective when the pH is low,51 and oxidative reactions arereaction type, this parameter was also measured as a potentialindicator of oil-in-water emulsion oxidation. From an initial averagevalue of 6.57, all samples tended to stabilize their pH at around 3.03after 18 days. The behavior of pH (shown in Fig. 3) was the oppositeof PV, the positive control and the tara Et_C3 sample being thosethat kept their pH stable for longer (up to 8 days above 6.30) before


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Figure 4. Peroxide value–pH regression for the oil-in-water emulsion oxidation (means of three replicates for eight different emulsions including six withtara extracts, positive control with Trolox and negative control) during 18 days. Continuous line relates to the regression equation pH = 6.80·e− 0.006·PV,with R2 = 0.9648.

decreasing. The Et_C2 (24 µg mL−1) pH also remained stable untilthat time, but decreased rapidly subsequently. In this case, ifinduction time was defined as the time in which the sample pHremained above 6, it was found to be 12 days for the Trolox controland the Et_C3 sample, 8 days for Et_C2, 7 days for Et_C1, 5 days forW_C2 and W_C1 and 2 days for the negative control. Therefore,for the most stable samples (Trolox control, Et_C3 and Et_C2), thisinduction time was the same as for the peroxide value, while forthe least stable ones pH began changing slightly later. Observingthis inverse relationship between the behavior of PV and thatdetected for pH, a regression was carried out on the means of thevalues for the eight different tested emulsions (two controls and sixtara pod extracts) during 18 days (Fig. 4). A negative exponentialrelationship was found between both parameters, the equationsbeing pH = 6.80·e− 0.006·PVand R2 = 0.9648.

The literature on lipid oxidation published in past years hasbeen extensive; however, some of the references that comparethe effect of various pH values on lipid oxidation are conflicting.50

Mancuso et al.52,53 reported that the initial oxidation of emulsionwas pH dependent, with a varying effect for different emulsifiers.They observed a higher oxidation rate at pH 7 than at pH 3,especially in oil-in-water emulsions stabilized with 1% Tween 20.The faster increase of primary oxidation products at higher pHcould be caused by increased formation or decreased degradationof these products, or by a combination of both factors. This low pHcan increase the solubility of iron and more iron can be partitionedinto the continuous phase, whereas at high pH insoluble ironprecipitates onto the emulsion droplet surface, and may increaselipid oxidation rates because of the close proximity of the ironand lipid substrate. Donnelly et al.54 reported that oxidationof a whey protein isolate (WPI)-stabilized emulsion decreasedwith decreasing pH (3–7) but in a Tween 20-stabilized emulsionoxidation increased with decreasing pH. Also Sorensen et al.55

reported that lipid oxidation increased when pH was decreasedfrom 6 to 3 in a 10% oil-in-water emulsion.

CONCLUSIONSTara (Caesalpinia spinosa) pods are an outstanding source of phe-nolic compounds and flavonoids, which can be extracted in differ-ent degrees using water or ethanol–water mixtures. The extractedamount of phenolics and flavonoids, as well as their antioxidantactivity, depend on both the solvent and the extraction method. In

this way, the use of a solution of ethanol 75% in a 1 h ultrasonic pro-cess results in the highest quantity of phenolics (0.4676 g g−1 DW)and provides the best antioxidant activity, as measured by ABTS+and ORAC methods (10.17 and 4.29 TE mmol L−1 g−1 DW, respec-tively). The highest amount of flavonoids is extracted with a 24 hmaceration in cold water, and 24 h cold maceration in ethanol 50%leads to the extract with the highest antioxidant activity assessedthrough the FRAP method. Tara extracts obtained with ethanol75% can be applied as antioxidants in oil-in-water emulsions. Theaddition of 48 µg mL−1 of this extract to the emulsion with 10%oil delays oxidation to the same extent as 17.8 µg mL−1 Trolox.The oxidative process of the emulsion can be assessed either bymeasuring the peroxide value or the pH, since both parametersare strongly related by an exponential-type equation.

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