J. of Supercritical Fluids 72 (2012) 168 175

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The Journal of Supercritical Fluids

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Extract ferleaves

Ma Teres tellEnrique Department of ationaP.O. Box 40, Pu

a r t i c l

Article history:Received 15 MReceived in reAccepted 26 Ju

Keywords:Mango leavesSupercritical FSubcritical WaCo-solventsMangiferinQuercetinAntioxidant activity

nd Swith b- anPa), n (COin, a

tioxidngo ltioxidgiferi

2012 Elsevier B.V. All rights reserved.

1. Introdu

The chamore friendof chemicalrening in oresources (pning, a typmatter intobiorenerycomponentas well as b

One of tof secondarthey are of gtical industrto comply chemical pr

SupercriExtraction eral advantmore selec

CorresponE-mail add

0896-8446/$ http://dx.doi.oction

llenges of this century based on a sustainable andly environment development have turned the vision

production toward a new industry concept of biomassrder to decrease rapid consumption of non-renewableetroleum, natural gas, coal, and minerals). In the begin-

ical biorenery convert essentially natural renewable bio-energy products. However, in the next generation, the feedstock will be fractionated further into valuables by extraction, fermentation and controlled pyrolysis,y more traditional methods.he rst stages in the new biorenery is the extractiony metabolites from low value biomass considering thatreater value in cosmetic, nutraceutical and pharmaceu-ies. The use of harmless extraction methods is essentialwith and environmental compatible and sustainableoduction [13].tical Fluid Extraction (SFE) and Subcritical Water(SWE) are interesting alternatives so present sev-ages including the use of green solvents, faster andtive processes, and the low degradation of chemical

ding author. Tel.: +34 956 016 579; fax: +34 956 016 411.ress: teresafernandez.ponce@uca.es (M.T. Fernndez-Ponce).

compounds [48]. Both techniques have been widely explored inrecent years in order to recover bioactive compounds from diverseplants and agri-industrial by-products [422].

Agricultural by-products of mango, particularly leaves and bark,present a high content on potent phenolic compounds, mainlymangiferin and quercetin, whose pharmaceuticals and nutraceu-tics properties have been demonstrated in several studies [2330].Mango is one of the most important tropical fruit worldwidewith a global production superior to 38 million tones and anarea harvested superior to 5 million hectares in 2010 [31]. Annu-ally pruning activity generates considerable quantities of residueswhich are usually burned or used for soil amelioration. Thus, con-version of pruning mango residues into valuable chemical productsby efcient and low impact extraction techniques results clearlyattractive within the concept of biorenery.

The extraction from mango by-products using SC-CO2 or sub-critical water has not been widely studied. Traditional solventextraction techniques are still usually used to recover bioactivecompounds from mango [23,2530] despite the drawbacks presentin these techniques [8,1113,16,18].

Mango leaves extracts with antioxidant activity have beenobtained by SC-CO2 extraction [32], but pure CO2, a nonpolar sol-vent, provide a low efciency to extract highly or slightly polarcompounds. Thus, the addition of CO2 modiers such as alcohol co-solvents should increase the extraction of polar polyphenols andalso improve the antioxidant activity of extracts, as described by

see front matter 2012 Elsevier B.V. All rights reserved.rg/10.1016/j.supu.2012.07.016ion of antioxidant compounds from difusing green technologies

a Fernndez-Ponce , Lourdes Casas, Casimiro ManMartnez de la Ossa

Chemical Engineering and Food Technology, Science Faculty, University of Cdiz Internerto Real 11510, Cdiz, Spain

e i n f o

ay 2012vised form 25 July 2012ly 2012

luid Extractionter Extraction

a b s t r a c t

Supercritical Fluid Extraction (SFE) aapplied in order to obtain extracts effects of extraction conditions on su(35 and 55 C), pressure (10 and 40 M(methanol/ethanol). The best conditiopared with SWE (4 MPa, 100 C, 10 g/mthan subcritical CO2 + ethanol. The ancation of the main polyphenols of mafor Kent variety, and extracts with anhigh content on the polyphenols man/ locate /supf lu

ent varieties of Mangifera indica

, Miguel Rodrguez,

l Agri-food Campus of Excellence, ceiA3,

ubcritical Water Extraction (SWE) from mango leaves werehigh phenolic content and potent antioxidant activity. Thed supercritical CO2 extraction were analyzed: temperature

percentage of co-solvent (0 and 20%) and type of co-solvent2 + 20% of ethanol at 10 MPa, 55 C, 20 g/min and 3 h) was com-nd 3 h) using seven mango cultivars. SWE was more efcientant activity was evaluated by DPPH assay, and the quanti-

eaves by HPLC analysis. SWE showed global yields up to 35%ant activities superior to (+)--tocopherol related with theirn and quercetin.

M.T. Fernndez-Ponce et al. / J. of Supercritical Fluids 72 (2012) 168 175 169

Fig

other authothe other hpounds, thiraw materia

Therefortion from mat differentExtracts weantioxidantperformancCO2 plus co

2. Materia

2.1. Materi

The seveington, Kenprovided byScientic Rein June 2010perature unlight.

Carbon S.A. (Barcradical (DP-d-glucosigrade (3,3(+)--tocopGermany). Tall HPLC grSpain). ThemilliQ grad

2.2. Extract

Extractiosupplied byschematic dFig. 1. This swith a thertwo pumpsbon dioxideregulator toto allow peextraction pwith approcyclonic sepin the extra

global yield (X0) for all extraction method was calculated consider-ing the ratio between mass of extract and mass of dry raw material.

A preliminary study was conducted in order to improve the yieldand antioxidant activity of the extracts obtained using CO2 and CO2

-solocesions:o-sonol a

20 gults w

preer, it

couldere c

this sed ion o

tioxi

ioxidpherined

ethodscrib0.1 mrds a

10

was ery 2once

calib

12, 7

perc2)

rem

EC50ted gple

tioxiAAI)

DPPws (

nal

na. 1. Schematic diagram of the high pressure equipment.

rs using different natural matters [7,8,1015,18]. Onand, although SWE is efcient to extract polar com-s technique has not been evaluated before using thisl.e, in this work Sub- and Supercritical Fluid Extrac-ango leaves using pure CO2 and CO2 plus co-solvents

conditions was studied and compared with SWE.re evaluated considering the global extraction yield, the

activity and the phenolic composition. In addition, thee of seven varieties of mango leaves was analyzed using-solvents and SWE.

ls and methods

als

n varieties of Mangifera indica L. leaves studied (Kens-t, Keitt, Tommy Atkins, Osteen, Ataulfo and Langra) were

Estacin Experimental La Mayora, Superior Centre ofsearch (CSIC), Mlaga, Spain. The leaves were collected

and February 2011. All leaves were dried at room tem-til constant weight and kept frozen in the absence of

dioxide (99.995%) was provided by Abello-Lindeelona, Spain). 2,2-Diphenyl-1-picrylhydrazyl, freePH), mangiferin (1,3,6,7-tetrahydroxyxanthone C2-de), quercetin 3--d-glucoside, purity 90% HPLC,4,5,7-pentahydroxyavone 3--d-glucoside), andherol were provided by SigmaAldrich (Steinheim,he organic solvents ethanol, methanol and acetic acid,adient grade, were provided by Panreac (Barcelona,

water used in all experiments was double-distillede.

ion procedure with solvents at high pressures

plus cotion prcondit55 C, cmetharate of

Resent onHowev100 Ctests w3 h.

Forwere uthe reg

2.3. An

Ant-tocodetermThe mods deAbout standaof a 6 DPPH and evDPPH cfrom a(1):

Abs = The

in Eq. (

%DPPH

Thecalculathe samThe anIndex (tion ofas follo

AAI =

The

n tests were carried out in a high pressure apparatus

Thar Technology (Pittsburgh, PA, USA, model SF100). Aiagram of the equipment used in this work is shown inet-up included an extraction vessel (capacity of 100 mL)mostatic jacket to control the extraction temperature,

with a maximum ow rate of 50 g/min (one for car- and the other for co-solvent), a back pressure valve

control the system pressure, and a cyclonic separatorriodic discharge of the extracted material during therocess. For all tests the extraction vessel was loaded

ximately 15 g of sample. Extracts were recovered in aarator and then collected in glass bottles and storedction solvent in darkness at 20 C prior to assay. The

concentratishowed pooidant activiactivity whAAI > 2.0 [3were compaquercetin 3

2.4. IdentiHPLC

Separatilent HPLC svents. The effects of different variables on the extrac-s were analyzed by considering the following operating

pressures of 10 and 40 MPa, temperatures of 35 andlvent percentages of 0 and 20% and type of co-solvent,nd ethanol. All tests were carried out with a CO2 ow/min and an extraction time of 3 h.ere compared with SWE. This technique is less depend-

ssure and highly dependent on temperature [19,20]. is important to consider that the temperatures above

generate unwanted oxidative processes [22], thus SWEarried out at 100 C, 4 MPa, a ow rate of 10 g/min and

preliminary study mango leaves of the variety Osteenas raw material so it is the variety widely cultivated inf Mlaga, Spain.

dant activity assay with DPPH

ant activity of extracts and standard compounds ((+)-ol, mangiferin and quercetin 3--d-glucoside) was

by 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay. employed was designed having in account the meth-ed by Brand-Williams and Scherer and Godoy [33,34].L aliquots of methanolic solutions of the samples ort different concentrations were each added to 3.9 mL5 mol/L DPPH methanolic solution. The absorbance ofmonitored spectrophotometrically at 515 nm at 0 min

min until the reaction reached the steady state. Thentration (CDPPH) in the reaction medium was calculatedration curve determined by linear regression with Eq.

09 CDPPH + 0.002 (1)entage of DPPH remaining was calculated as described

aining = CDPPHtCDPPH0

100 (2)

(efcient concentration providing 50% inhibition) wasraphically using a non-linear tting curve by plottingconcentration vs. the % DPPH remaining on steady state.dant activity was expressed as the Antioxidant Activity

which was calculated considering the nal concentra-H and the EC50 of the tested compound in the reactionEq. (3)):

concentration of DPPH (g/mL)EC50 (g/mL)

(3)

l concentration of DPPH was calculated respect to theon of DPPH in the reaction medium. Plant extractsr antioxidant activity when AAI < 0.5, moderate antiox-ty when AAI is between 0.5 and 1.0, strong antioxidanten AAI is between 1.0 and 2.0, and very strong when4]. The assays were carried out in triplicate. Resultsred with standards of (+)--tocopherol, mangiferin and--d-glucoside.

cation and quantication of phenolic compounds by

on of phenolic compounds was performed using an Agi-eries 1100 system (Agilent, Germany) equipped with a

170 M.T. Fernndez-Ponce et al. / J. of Supercritical Fluids 72 (2012) 168 175

quaternary pump, an autosampler, a 250 mm 4.6 mm i.d., 5 m,C18 reversed-phase column (Thermo Electron Corporation) and aUV/vis detector, connected to a HP ChemStation software.

The method used is a modication of the method described byBarreto et awere A (aceconditions gradient 5gradient 25gradient 32and recondlinear gradilinear gradiwas 0.9 ml/was operate

Phenolictimes and sponding stpolyphenol3--d-gluco

A = 54, 252A = 87, 077where A is expressed iboth calibracarried outmeasuremetwo varietie

2.5. Experim

A multidetermine variables) ois used as sbest CO2 mofor tempertion procesusing 22 + 2adjusted. Ocarried out tion yield edata were acal Graphicto predict tyield of the

2.6. Evalua

Mango isworldwide tivars usedimportanceand Keitt arexported byPakistan, Brtivar of Mexis the essencommerciaFinally, Kenreceived in

The perf(Kensingtongra) was e

lobalsteen

inaryPa, 5

C, 4ltivantio

ults and discussion

percritical Fluid Extraction with pure CO2

of the advantages of SFE is CO2 solvating power can beulated by changing pressure (P) and/or temperature (T);re, knowing the inuence of extraction conditions on thes is necessary to obtain high extraction yields. SC-CO2 extrac-lds obtained at different conditions of P and/or T from mango

of the variety Osteen are shown in Fig. 2A. Global yieldsed with pure CO2 were unsatisfactory and even changes inraction conditions led to negligible improvements. The high-ld was 1.22 0.13% obtained at 40 MPa and 55 C.

effect of pressure on SC-CO2 extraction from mango leavessitive for both temperatures studied. This behavior can beed by the increase in density with pressure resulting in

r solvating power of CO2 [16,17]. Moreover, high pressuresdisruptions in plant cells and allow compounds to be morele, thereby favoring the extraction yield [37].ut the effect of temperature, it is more complex because it

ds on two factors. The density of CO2 decreases with tem-re, reducing the solvating power, while the solute vaporre increases favoring the solubility on SC-CO2. Thus, depend-

the operating conditions, one factor is dominant over the[16,17]. In this way, at low pressures (10 MPa) the effect

density was predominant, so high extraction yields wereed on reducing temperature. But, at high pressures (40 MPa)l. [23]. The solvents that constituted the mobile phasetic acidwater, 2:98, v/v) and B (methanol). The elutionapplied were: 02 min, 5% B isocratic; 27 min, linear25% B; 711 min, 25% B isocratic; 1119 min, linear32% B; 1927 min, 32% B isocratic; 2728 min, linear40% B; 2838 min, 40% B isocratic and nally, washingitioning steps of the column were included (3850 min,ent 40100% B; 5060 min, 100% B isocratic; 6070 min,ent 1005% B; and 5 min, 5% B isocratic). The ow-ratemin and the injection volume was 50 L. The systemd at room temperature.

compounds were detected at 340 nm by its retentionquantied using a calibration curve of the corre-andard compounds. The calibration curve of the mains of mango leaves, mangiferin (Eq. (4)) and quercetinside (Eq. (5)), was as follows:

C 100.12 (4) C 130.11 (5)the area expressed in mAu and C is the concentrationn g/ml. The correlation coefcient (R2) was 0.9999 fortion curves. The experiments on each extraction were

in triplicate in order to evaluate the variability of thents. HPLC chromatograms of the extracts obtained fors studied are shown in Fig. 5.

ental design

level factorial design was carried out in order tothe effect of temperature and pressure (experimentaln the yield of the process when CO2 + 20% of ethanololvent system, accordingly ethanol was selected as thedier. The ranges for the factorial design were 3555 C

ature, and 1040 MPa for pressure. Then, the extrac-s was analyzed through a factorial experimental design

central points where temperature and pressure weren the basis of this design a total of 6 experiments werein a single block. The response variable was the extrac-xpressed as g/100 g of dry matter. The experimentalnalyzed by Statgraphics Plus 5.1 (19942001, Statisti-s Corp.). Empirical correlations were developed in orderhe inuence of extraction conditions on the extraction

process studied.

tion of different mango leaves cultivars

an Indian native fruit that occupies the third position inproduction and importation of tropical fruits. The cul-

in the present work were selected according to their worldwide. The Florida cultivars Tommy Atkins, Kente the mainly current commercial varieties produced and

most countries, including leading exporters as Mexico,azil, Peru and Ecuador. Ataulfo is other important cul-ico, which is too appreciated in North America. Osteential cultivar of Spain production. Langra is an importantl mango variety of north India with good quality fruits.sington is the dominant variety grown in Australia welloverseas markets [35,36].ormance of the M. indica L. varieties above mentioned, Kent, Keitt, Tommy Atkins, Osteen, Ataulfo and Lan-valuated using the best conditions obtained in the

Fig. 2. Gvariety O

prelimat 10 Mat 1003 h. Cuyield, a

3. Res

3.1. Su

Onemaniptherefoprocestion yieleavesobtainthe extest yie

Thewas poexplaingreatecause availab

Abodepenperatupressuing onother on theobtain yield (A) and antioxidant activity (B) of mango leaves extracts of the obtained using SC-CO2, CO2 + 20% methanol and CO2 + 20% ethanol.

study with Osteen variety: CO2 + 20% (w/w) of ethanol5 C, 20 g/min and 3 h, and also using subcritical water

MPa, a ow rate of 10 g/min, and an extraction time ofrs were evaluated accordingly to the global extractionxidant activity and phenolic prole.

M.T. Fernndez-Ponce et al. / J. of Supercritical Fluids 72 (2012) 168 175 171

Table 1Extraction yields of phenolic compounds obtained from mango leaves of the variety Osteen, expressed as mg/100 g dry leaves.

Extraction method Conditions Yield of phenolic compoundsa (mg/100 g dry leaves)

MangiferinR

Quercetin

SC-CO2 n

CO2 + 20% m 7971

CO2 + 20% et 1164

Subcritical w 1

a Yield of ph valueb n.d.: comp

the effect oof the solut

In spite and high tehigh yields content onpoorly solupolyphenolglucoside kn

Table 1 sby HPLC forshows the Mangiferin HPLC on SCpounds henresult, the aantioxidantCO2 at low did not leadresulted ina

3.2. Subcrit

Given thpounds fronecessary tpolar subst[1014,38the solvent

Among atrile, acetonand ethanohol co-solvbonding wiinteractions[18,40].

Methanoble up to 2this is morebut the temhigher and ethanol, it sand may beused in nutmind, ethanwork.

extrhanosted,ecau

poinse syefciconced thocyaentstratipom

reasand ing t

augmion o

globed wld obentsl alloO2 at

inu mixserve wi. The in100400 bar 3555 C

ethanol 100 bar 35 C 55 C

400 bar 35 C 55 C

hanol 100 bar 35 C 55 C

400 bar 35 C 55 C

ater 40 bar 100 C

enolic compounds expressed as mg/100 g dry matter was represented as the meanound not detected.

f vapor pressure was dominant and the high volatilityes resulted in an overall increase in the extraction yield.of the increase on the global yield at high pressuremperature, SC-CO2 was not efcient enough to obtainfrom mango leaves. This can be attributing to the high

polar phenolic compounds of mango leaves [23,24],ble in SC-CO2, a nonpolar solvent [1014,38]. The mains of mango leaves are mangiferin and quercetin 3--d-own by their potent antioxidant properties [24,2630].

hows the extraction yields of both compounds obtained the different extraction techniques explored, and Fig. 2Bantioxidant activity of extracts obtained with SC-CO2.and quercetin 3--d-glucoside were not detected by-CO2 extracts. Both polyphenols are slightly polar com-ce SC-CO2 is not efcient to extract them [38]. As antioxidant activity of SC-CO2 extracts was poor too. Any

activity was observed for the extracts obtained with SC-pressures (10 MPa), and raising the pressure to 40 MPa

to an increase in this activity. Consequently, pure CO2ppropriate to obtain extracts from mango leaves.

ical Fluid Extraction with CO2 plus co-solvents

e low capacity of SC-CO2 to extract antioxidant com-m mango leaves, the addition of co-solvents waso improve the quality of extracts. The solubility ofances, such as polyphenols, in SC-CO2 is very low40], however the use of organic co-solvents increases

power of CO2 and the extraction yield [7,18,3840].ll the modiers including methanol, ethanol, acetoni-

Theof metture tepoint bcritical

Themore lower reportof anthco-solvconcenpeach

Theof CO2increasdue toextract

Theobtainthe yieco-solvethanopure C

Thecriticalthat obincreas10 MPaincrease, water, ethyl ether and dichloromethane, methanoll are most frequently used for SFE of polyphenols. Alco-ents induce dipole/dipole interactions and hydrogenth polar functional groups, and also they can break polar

solutematrix increasing the solubility of polar solutes

l is commonly used as co-solvent because it is misci-0% with CO2 and some publications have shown that

efcient than ethanol to remove polyphenols [40,41],perature necessary to reach the supercritical state iscould be not suitable for natural products [18]. Aboutignicantly enhanced the extraction of avonoids [18]

a better choice considering it as non-toxic and can beraceutical or cosmetic applications [39]. With this inol and methanol were used as CO2 modiers in this

solvent imintensicatat 40 MPa tmango leav

In relatisure had aco-solventsincrease in decreased pressures eextracts wibetter antioco-solvents55 C (6.53

The negusing CO2 +t = 20.9 min 3--d-glucosideRt = 34.4 min

.d.b n.d.

.8 0.1 6.1 0.13.9 0.1 88.7 1.0.8 0.6 6.4 0.12.9 0.3 15.3 0.191.8 0.3 140.9 1.284.5 0.4 231.4 3.32.6 0.2 55.4 0.10.6 0.0 49.7 0.4365.9 1.2 409.5 6.7

standard deviations.

actions with co-solvents were carried out using 20%l/ethanol. At the conditions of pressure and tempera-

the mixtures of CO2 + co-solvent are below their criticalse high concentrations of CO2 modiers increase thet of the mixture [7,39].stems called enhanced uidity liquids have resultedent to extract polar compounds than mixtures withentrations of co-solvents. For example, other authorsat 20% of co-solvents duplicate the extraction yieldsnins from red grape pomace instead using only 5% of

[7]. Adil et al. [39] showed that the optimum ethanolons for CO2 extraction of polyphenols from apple andace were found to be 20%.on for this is based on the fact that the solvating powerthe solubility of polar compounds in CO2 increase withhe amount of co-solvent concentration from 5 to 30%,ented phenolalcohol interactions that facilitate the

f the solute [7,38,39].al extraction yields and antioxidant activity of extractsith both solvent systems are shown in Fig. 2. Comparingtained using SC-CO2 and the mixtures of CO2 + 20% of

(Fig. 2A), it is clear that the addition of methanol orwed higher extraction yields than those obtained with

all conditions tested.ence of P and/or T on the extraction process using sub-tures of CO2 plus co-solvents resulted different thaned with pure CO2. About temperature, the global yieldsth temperature when extractions were carried out atis positive effect of temperature is related with the

the diffusivity and decrease in the viscosity of the

proving the mass transfer properties along with theion of solute volatility favoring the extraction. However,he effect of temperature on the extraction process fromes was not relevant using co-solvents.on to the pressure, at low temperatures (35 C), pres-

positive effect on the extraction yields using both. By contrast, at high temperatures (55 C) a markedthe extraction yield was observed when pressure wasfrom 40 to 10 MPa, showing a negative effect. Lownhanced the extraction of polyphenols resulting in

th higher content on mangiferin and quercetin and thusxidant capacity. The higher extraction yields for both

(methanol and ethanol) were obtained at 10 MPa and 0.83 and 6.37 0.13%, respectively).ative effect of pressure on the extraction process

20% of co-solvent was also observed for the phenolic

172 M.T. Fernndez-Ponce et al. / J. of Supercritical Fluids 72 (2012) 168 175

extraction yield. The highest yields of phenolic compounds wereobtained at 10 MPa and 55 C (184.5 0.4 and 231.4 3.3 mg/100 gdry leaves for mangiferin and quercetin, respectively) (see Table 1).This negative effect of pressure has been also described for otherraw materiatent was oblueberry aof anthocymethanol/wthat at 15 Mfrom grape reported a nolic conten

A scientnot describthat lower p[7,14,15,39ciently enhasuch as aloeMoreover, iglobal yieldto bioactiveextraction pof matrix an

The globas co-solvenof 0.95, notactivity of eyields of thquercetin 3in Table 1.

The addinolic compinteractionswith phenoever, the hiCO2 + 20% oextract phe

The greglucoside oethanol allactivities thence of P obtained wto the effec

Mango lemethanol/eity with va(10 MPa) anwith a stroand a modebecause lonols resultiquercetin a

Consequmango leav(10 MPa), her. Furtherconsidered and food in

3.2.1. AnalyResults

analyzed bylyze the in

areto diagram for the global extraction yield obtained with CO2 + 20% ofusing mango leaves of the variety Osteen.

Paren in

nge ownesencanf 0.95ive ostionpiricnd the varcesslysisnd thctorss usinmpeas thxtrac

em give

.675

al ex pres9961

bcrit

pite oangPa

y is ocoption yh aned usereds from

glo 1.1antlO2 p

d effects and analysis of variance for the solvent system CO2 + 20% ethanol.

le Effects p-Value

0.69 0.03842.17 0.0041

2.18 0.0041ure; T: temperature; PT: pressure/temperature.ls. Laroze et al. [15] reported that highest phenolic con-btained at low pressure, 7.515 MPa from cranberry,nd raspberry. Mantell et al. [7] described an increaseanins extraction from grape marc using CO2 + 20%ater at low pressures. Vatai et al. [14] also observedPa higher amounts of total phenols were extracted

marc using CO2 plus co-solvents. Finally, Adil et al. [39]negative effect of pressure below 50 MPa on total phe-t of apple and peach pomace using enhanced uidities.

ic explanation for this behavior is not clear and it ised in the literature. The above mentioned cases showressures improve the extraction yield using co-solvents]. But other authors reported that high pressures ef-nced uidity extractions using different raw materials,

vera leaf skin, chamomile, grape seed or marigold [18].n some cases the modier has little or no effect on the

but improves the extract composition with respect compounds. Therefore, the effect of pressure on therocess using co-solvents possibly depends on the typed the interactions solutematrix.al extraction yields obtained using methanol or ethanolt showed no signicant differences at a condence levelwithstanding the phenolic yields and the antioxidantxtracts showed remarkable differences. The extractione main polyphenols of mango leaves (mangiferin and--d-glucoside) using CO2 plus co-solvents are shown

tion of methanol or ethanol helps the extraction of phe-ounds because both co-solvents are capable of strong

(hydrogen-bonding and dipoledipole interactions)ls facilitating the extraction of these compounds. How-ghest quantities of polyphenols were recovered usingf ethanol showing this co-solvent is more efcient tonolic compounds from mango leaves.ater content on mangiferin and quercetin 3--d-f mango leaves extracts obtained with CO2 + 20% ofows extracts to be obtained with better antioxidantan CO2 + 20% of methanol extracts (Fig. 2B). The inu-and/or T on the antioxidant activity of the extractsith CO2 + 20% (w/w) methanol/ethanol was analogoust described for the global yields.aves extracts obtained at high pressures (40 MPa) usingthanol as co-solvent presented a poor antioxidant activ-lues of AAI lower than 0.5. By contrast, low pressuresd high temperatures (55 C) led to obtained extracts

ng AA (superior than 1.0) using ethanol as co-solvent,rate AA using methanol. These high activities occurredwer pressures enhanced the extraction of polyphe-ng in extracts with higher content on mangiferin andnd thus better antioxidant capacity.ently, the extraction of antioxidant compounds fromes is more advantageous on working at low pressureigh temperature (55 C) and with ethanol as CO2 modi-more, ethanol provides an additional advantage as it isto be a green solvent whose use in the pharmaceuticaldustries is not restricted.

sis of the experimental designobtained with CO2 + 20% of ethanol were statistically

a multifactorial experimental design in order to ana-uence of the variables on the process.

Fig. 3. Pethanol

Theis showthe raalso shis repra signilevel oa positin que

Emdata alink ththe pro

Anature athe faprocessure/tewherehigh eture.

Theyield is

Y = 8Y: glob(C); P:was 0.

3.3. Su

In sfrom mat 10 Mactivit(+)--textractoo higexplorconsidpound

The(24.24signicwith C

Table 2Estimate

Variab

PT PT

P: pressto diagram for the global yield as the response variable Fig. 3. The estimated effects and interactions betweenof variables studied and the analysis of variance are

in Table 2. The degree of signicance of each factorted by its p-value; factors with a p-value < 0.05 havingt inuence on the extraction process for a condence. The sign associated with each of the effects indicatesr negative inuence on the yield caused by the variable.al correlations were obtained using the experimentale program Statgraphics Plus 5.1. These correlationsiables with inuence on the global extraction yield of

using CO2 + 20% ethanol as solvent system. of Table 2 and Fig. 3 indicates that the tempera-e combined interaction of pressure/temperature are

that inuence the global extraction yield of theg CO2 + 20% ethanol. The combined interaction of pres-rature showed a negative effect on the extraction yielde effect of temperature was positive. This explains thetion yield obtained at low pressure and high tempera-

pirical correlation obtained for the global extractionn below (Eq. (6))

8 + 0.0304 P + 0.290167 T 0.000726667 P T (6)traction yield (g/100 g of dry matter); T: temperaturesure (MPa). The resulting determination coefcient (R2).

ical Water Extraction

f the strong antioxidant activity of the extracts obtainedo leaves of the variety Osteen using CO2 + 20% of ethanoland 55 C (1.23 0.01 DPPH g/g dry extract), suchstill far from that AAI obtained for the antioxidantherol (3.84 DPPH g/g dry extract). Moreover, theields obtained with this solvent system were also notd are susceptible to be improved. In this way, SWE wasing mango leaves as raw material so this technique is

highly efcient to extract polar or slightly polar com- different natural matters [1922].bal yield of the process obtained with SWE7%) from mango leaves of the variety Osteen wasy superior to those obtained with SC-CO2 and evenlus co-solvents (see Fig. 2A). As well, the high efciency

M.T. Fernndez-Ponce et al. / J. of Supercritical Fluids 72 (2012) 168 175 173

Fig. 4. GlobalCO2, 3 h) and SKensington; 2

of subcriticdemonstratquercetin (respectively

The highbe explainesolvent notbe reducedhydrogen bpounds, as conditions water [19,2

On the leaves of thity (7.92 0stronger thwas superioactivity of tdry extract)the high an

In generwith their ppounds andthe antioxidthe subcritiwith their hbut also thtributing in

The highto obtain exgreen extraantioxidantsupercritica

3.4. Evalua

The studTommy Atkcomparisonactivity of tconditions ow rate of100 C, 4 MP

The globstudied areobtained wstudied. The

SWEb

Man

gife

rin

Rt=

20.9

min

Quer

cetin

3--d

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Rt=

34.4

min

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idan

tac

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tyM

angi

ferin

Rt=

20.9

min

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cetin

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coside

Rt=

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min

Yie

ld

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t)

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ld

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(g/1

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0.23

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0.01

0.05

1.3E

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83

0.02

6.61

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0.01

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6.9E

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0.02

0.58

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4

7.25

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1.37

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6.76

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1.64

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ns

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ined

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the pre

lim

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y

study

(100

bar,

55 C

, 20

g/m

in

of

CO

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d

3

h).

d

3

h.

PH

g/g

dry

extr

act

was

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sente

d

as

the

mea

n

valu

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iation

s.

g/10

0

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dry

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as

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d

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iation

s. yields obtained with CO2 + 20% ethanol (10 MPa, 55 C, 20 g/min ofWE (4 MPa, 100 C, 10 g/min, 3 h) from different mango cultivars: 1,

, Kent; 3, Keitt; 4, Tommy Atkins; 5, Osteen; 6, Ataulfo; 7, Langra.

al water to extract phenolic compounds was alsoed with the high recovery quantities of mangiferin and1365.9 1.2 and 409.54 6.7 mg/100 g of dry sample,) (see Table 1).

extraction of polyphenols with subcritical water cand because of the fact that although water is a too polar

appropriate to extract polyphenols, its polarity can with increasing temperature due to a reduction of itsonding propensity. In consequence, slightly polar com-polyphenols, with low solubility in water at ambientor in SC-CO2, can be much more soluble in subcritical0].other hand, subcritical water extracts from mangoe variety Osteen present a potent antioxidant activ-.16 DPPH g/g dry extract) (see Table 3) signicantlyan those obtained with CO2 plus ethanol. This activityr than (+)--tocopherol, and similar to the antioxidanthe standard compounds mangiferin (7.80 DPPH g/g

and quercetin (7.06 DPPH g/g dry extract), showingtioxidant capacity of subcritical water extracts.al, the antioxidant activity of extracts can be correlatedhenolic content. Nonetheless, the activity of each com-

the content of each one in the extract can inuence onant activity too. So, the potent antioxidant capacity ofcal water extracts of the variety Osteen may be relatedigher content on mangiferin than quercetin (Table 3),

e co-extraction of other antioxidant compounds con- the activity of extracts.

efciency of SWE to recover phenolic compounds andtracts with potent antioxidant activity shows that thisction technology is a promising alternative to extract

compounds from mango leaves more efciently than ts

obta

ined

with

CO

2+

20%

ethan

ol

and

Subc

ritica

l Wat

er

Extr

action

from

diffe

rent m

ango

cultiv

ars.l CO2 extraction.

tion of mango cultivars

y of seven mango varieties (Kensington, Kent, Keitt,ins, Osteen, Ataulfo and Langra) was carried out by

of extraction yields, phenolic prole and antioxidanthe extracts obtained using CO2 + 20% ethanol at the bestobtained from the preliminary study (10 MPa, 55 C, a

20 g/min and 3 h), and also using subcritical water ata, 10 g/min and 3 h.al yields obtained with SWE from the seven varieties

shown in Fig. 4. SWE yields are higher than thoseith CO2 + 20% ethanol for the entire mango cultivars

best global yields were obtained for the varieties Kent, Table

3Antiox

idan

t

activi

ty

and

phen

olic

conte

nt

of

extr

ac

Man

go

cultiv

ars

CO

2+

20%

ethan

ola

Antiox

idan

tac

tivi

ty

AAIc

(CDPP

H/E

C50

)

Ken

singt

on2.

13

0.24

Ken

t

2.16

0.08

Kei

tt0.

78

0.05

Tom

my

Atk

ins

2.07

0.01

Ost

een

1.09

0.01

Ata

ulfo

1.59

0.04

Langr

a

2.74

0.01

Stan

dar

d

com

pounds

(+)--T

ocop

her

ol

3.65

0.01

Man

gife

rin

-d

-glu

coside

7.80

0.02

Quer

cetin

3--d

-glu

coside

7.06

0.01

aCO

2+

20%

ethan

ol

was

oper

ated

at

best

conditio

bSW

E

was

oper

ated

at

40

bar,

100

C, 1

0

g/m

in

anc

AAI,

Antiox

idan

t

Act

ivity

Index

expre

ssed

as

DP

dYie

ld

for

each

phen

olic

com

pou

nd

expre

ssed

ase

Con

tent

of

phen

olic

com

pou

nd

expre

ssed

as

g/1

174 M.T. Fernndez-Ponce et al. / J. of Supercritical Fluids 72 (2012) 168 175

Fig. 5. HPLC c ) and SCO2 + 20% etha C, 10 g

Keitt, Tomm33.71 1.12varieties La(27.84 0.6

When COeties Tomm(8.15 0.31Keitt, Kensiever, globalthose obtai

The extrleaves (manby HPLC. Thdry matter critical watof extracts o

The yield3--d-glucoobtained wvarieties Atlic compounusing subcrilar to the traditional svarieties (3of mangiferues were oband 0.50 be a good acompounds

About tshows thatto mangifecient than Cwater extra

hestaves19.1 ed w

and 3.3Et systes (0hromatograms of extracts obtained from Osteen variety using CO2 + 20% ethanol (Anol was carried out at 10 MPa, 55 C, 20 g/min of CO2, 3 h; and SWE at 4 MPa, 100

y Atkins and Kensington (35.42 0.53, 34.17 0.73, and 33.34 2.04%, respectively) using SWE, while thengra, Ataulfo and Osteen presented lower global yields0, 26.96 1.00 and 24.24 1.17%, respectively).2 + 20% of ethanol was used as solvent system, the vari-y Atkins and Kent presented the highest global yields

and 8.06 0.15%, respectively) followed by Langra,ngton and Osteen (6.37 0.18 to 7.37 0.22%). How-

the higdry leety (1obtainAtkins0.23 solvenvarieti yields obtained with CO2 extraction were inferior thanned using subcritical water.action yields of the main polyphenols present in mangogiferin and quercetin 3--d-glucoside) were analyzede yields of the phenolic compounds in terms of mg/100 gfor extracts obtained with CO2 + 20% ethanol and sub-er are shown in Fig. 5, and typical HPLC chromatogramsbtained with both solvent systems are shown in Fig. 5.s of the phenolic compounds mangiferin and quercetinside obtained with SWE were higher than thoseith CO2 + 20% of ethanol (Table 3). For mangiferin, theaulfo and Langra allowed high recoveries of this pheno-d (3.37 2.1E3 and 3.09 9.1E3 g/100 g dry leaves)itical water as solvent system. These results were sim-yield of mangiferin reported by other authors usingolvent extraction methods from different mango leaves.719.36 g/100 g dry leaves) [23]. By contrast, the yieldsin obtained with CO2 + 20% were inferior. The best val-tained for the varieties Kent and Langra (0.58 5.2E44.6E4 g/100 g dry leaves). Consequently, SWE wouldlternative to traditional methods to extract phenolic

from mango leaves.he extraction of quercetin 3--d-glucoside, Table 3

the recovery of this phenolic compound was inferiorrin. However, SWE has proved to be also more ef-O2 + 20% ethanol to extract this polyphenol. Subcriticalcts from varieties Ataulfo, Osteen and Keitt showed

The antobtained frobtained usantioxidantpresented textract) cloington, Tompresented sthan 1.0 g

By contrdant activitextract) andcases the coextracts waextracts (se

In this wextracts (4.lic compouto CO2 + ethon mangifeantioxidantcontribute

4. Conclus

The resualternative WE (B): 1, mangiferin; 2, quercetin 3--d-glucoside. Extraction with/min, 3 h.

quercetin yields (0.41 6.7E3 to 0.42 6.1E3 g/100 g) while the lowest yield was for Kensington vari- 0.1 mg/100 g dry matter). In relation to the yieldsith CO2 + 20% ethanol, the varieties Kent, Keitt, Tommy

Osteen showed similar yields (0.11 1.2E3 to3 g/100 g dry leaves), and the lowest yields using thisem were obtained from Kensington, Ataulfo and Langra.04 6.6E4 to 0.05 1.3E3 g/100 g dry leaves).

ioxidant activities and phenolic content of extractsom the seven varieties are shown in Table 3. Extractsing CO2 + 20% of ethanol as solvent system showed good

activities. Using this solvent system Langra extractshe highest activity prole (2.74 0.01 DPPH g/g dryse to (+)--tocopherol activity, and the cultivars Kens-my Atkins, Kent, Kensington, Ataulfo and Osteen

trong antioxidant activities with AAI values superior/g dry extract.ast, subcritical water extracts showed a potent antioxi-y up to (+)--tocopherol activity (3.65 DPPH g/g dry

superior to CO2 + ethanol extracts even though in somentent on mangiferin and quercetin 3--d-glucoside ofs similar than the phenolic content of CO2 + ethanole Table 3).ay, the potent antioxidant activity of subcritical water

027.92 DPPH g/g dry extract), similar to the pheno-nds mangiferin and quercetin and signicantly superioranol extracts, can be attributed to the high contentrin and quercetin, but also the presence of other polar

compounds, not identied in this work, which alsoto their antioxidant activity.

ions

lts presented in this study show that SWE is an excellentto recover high quantities of phenolic compounds from

M.T. Fernndez-Ponce et al. / J. of Supercritical Fluids 72 (2012) 168 175 175

mango leaves thanks to the high extraction yields and the potentantioxidant activity of extracts obtained by this extraction method.SWE resulted more efcient than SC-CO2 extraction even thoughthe performance of CO2 extraction was improved with the addi-tion of ethanol as CO2 modier. Quality of subcritical water extractswas signicantly superior to that obtained with the subcritical mix-tures of CO2 + 20% of ethanol in terms of phenolic content and alsoantioxidant activity. Subcritical water extracts from the varietyOsteen presother varieting mango lwith potenindustries. from mangaccount the

Acknowled

The autthe Spanish22974), whresearch groSpain) for pzalez Fern

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Extraction of antioxidant compounds from different varieties of Mangifera indica leaves using green technologies1 Introduction2 Materials and methods2.1 Materials2.2 Extraction procedure with solvents at high pressures2.3 Antioxidant activity assay with DPPH2.4 Identification and quantification of phenolic compounds by HPLC2.5 Experimental design2.6 Evaluation of different mango leaves cultivars

3 Results and discussion3.1 Supercritical Fluid Extraction with pure CO23.2 Subcritical Fluid Extraction with CO2 plus co-solvents3.2.1 Analysis of the experimental design

3.3 Subcritical Water Extraction3.4 Evaluation of mango cultivars

4 ConclusionsAcknowledgmentsReferences