Inclusion complexes of β-cyclodextrin with chlorogenic acids (CHAs) from crude and purified aqueous extracts of green Robusta coffee beans (Coffea canephora L.)

Food Research International xxx (2013) xxx–xxx

FRIN-04877; No of Pages 12

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Inclusion complexes of β-cyclodextrin with chlorogenic acids (CHAs)from crude and purified aqueous extracts of green Robustacoffee beans (Coffea canephora L.)

Grażyna Budryn a,⁎, Ewa Nebesny a, Bartłomiej Pałecz b, Danuta Rachwał-Rosiak a, Paweł Hodurek c,Karolina Miśkiewicz a, Joanna Oracz a, Dorota Żyżelewicz a

a Institute of Chemical Food Technology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 90-924 Lodz, Polandb Departments of Physical Chemistry, Faculty of Chemistry, University of Lodz, 90-131, Lodz, Polandc Institute of Economic and Social Policy, Krosno State College, 38-400 Krosno, Poland

⁎ Corresponding author at: Institute of ChemicalBiotechnology and Food Sciences, Lodz University of T42 6313458.

E-mail address: [email protected] (G. Budryn)

0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.foodres.2013.10.013

Please cite this article as: Budryn, G., et al., Incextracts of green Robusta coffee ..., Food Rese

a b s t r a c t
a r t i c l e i n f o
Article history:Received 20 August 2013Received in revised form 3 October 2013Accepted 7 October 2013Available online xxxx

Keywords:Green coffee extractβ-cyclodextrinInclusion complexesCentrifugal partition chromatography (CPC)LC–MS/MS

The initial aim of the study was to purify the crude aqueous coffee bean extract, containing CHAs and caffeinewith the use of the centrifugal partition chromatography (CPC) technique to obtain a higher concentration ofCHAs with a simultaneous decrease of caffeine quantity. As a result of extract purification CHA content wasincreased by about 50% and caffeine was completely eliminated. CHAs were identified by the LC-ESI–MS/MStechnique. A further aim was to obtain inclusion complexes of both crude and purified extracts withβ-cyclodextrin (β-CD). The highest obtained concentration of CHAs in a complex with β-CD amounted to19 g/100 g. The formation of inclusion complexes was confirmed with the use of ESI-MS/MS and differentialscanning calorimetry (DSC) techniques. The analysis of antioxidant capacity of inclusion complexes with β-CDrevealed that crude and purified extracts showed about 2-fold higher antioxidant capacity than the obtainedcomplex.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The term “chlorogenic acids” is used to refer to a family of transhydroxycinnamic acids, one of the most important groups of phenoliccompounds, occurring abundantly in many plants. These catechol-likecompounds have a broad range of beneficial biological activities suchas anti-bacterial (Lou, Wang, Zhu, Ma, & Wang, 2011.), anti-fungal(Sung & Lee, 2010), hepatoprotective (Shi et al., 2013), anti-thrombotic(Satake, Kamiya, An, Oishi, & Yamamoto, 2007), anti-inflammatory (El-Medany, Bassiouni, Khattab, & Mahesar, 2011), hypoglycemic (Bassoliet al., 2008) and antioxidant (Sato et al., 2011). The last propertyreduces the risk of several oxidative stress-related diseases, includingatherosclerosis (Cheng, Dai, Zhou, Yang, & Liu, 2007), cancer (Lee &Lee, 2006) and Alzheimer's disease (Silva, Ferreres, Malva, & Dias,2005). A few plants accumulate chlorogenic acids in quantitiessufficient to have physiological effects (Kim, Shang, & Um, 2010). Oneof them is coffee, whose green beans contain 4–10% of these com-pounds on fresh basis. Relatively high concentrations of CHAs in coffeebeans and additionally large amounts of coffee processed in the whole

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lusion complexes of β-cyclodarch International (2013), htt

world make green coffee beans one of the richest and most readilyavailable sources of chlorogenic acids (Moon, Yoo, & Shibamoto, 2009).

High antioxidant capacity is observed even in poor quality beans(crude and defective), containing similar amounts of CHAs as highquality beans, however they are eliminated before roasting due totheir ability to generate undesirable aroma (Ramalakshmi, Kubra, &Rao, 2008). Because of high concentration of CHAs in coffee beansand their good solubility in water it is possible to obtain even crudepreparations with high quantities of these compounds. These kinds ofextracts can be used as food components and food supplements. Unlikepreparations containing phenolic compounds from fruits, vegetables,legumes or nuts they also contain caffeine, which additionallystimulates the central nervous system. Some consumers, howeversuffer from overstimulation, resulting in increased heart rate anddifficulty in sleeping or stomach irritation even after consumption ofrelatively small amounts of caffeine (Weiss et al., 2010). Therefore,for this group of consumers it would be beneficial to purify crudeextracts of green coffee, leading to increase of CHA concentration andsimultaneous decrease of caffeine content. To meet this requirementpurification with the use of CPC seems to be most promising (http://www.kromaton.com/en/the-cpc/technologies). It is a useful techniquefor separation of a mixture of compounds with different partitioncoefficients in a two-phase solvent system. Previously, 5-O-caffeoylquinic acid (5-CQA), the most abundant phenolic compound

extrinwith chlorogenic acids (CHAs) from crude and purified aqueousp://dx.doi.org/10.1016/j.foodres.2013.10.013

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Table 1Partition coefficient (K) values of 5-O-caffeoylquinic acid in two-phase solvent systems.

Solvent system Volume ratio Partition coefficient (K)

Water+1% TFA–n-butanol–ethyl acetate 1:1:0 0.015:4:1 0.315:3:2 0.365:2:3 0.395:1.5:3.5 1.375:1:4 2.391:0:1 0.33

Water+1% TFA–ethanol–ethyl acetate 5:2:3 3.265:1:4 1.21

Water–ethanol–ethyl acetate 5:2:3 1.735:1:4 1.17

TFA— trifluoroacetic acid.

2 G. Budryn et al. / Food Research International xxx (2013) xxx–xxx

in green coffee beans, has been purified by CPC in extracts from otherplants, including honeysuckle (Wang, Jiang, Yang, & Wu, 2008) andeucommia (Li et al., 2007). A salting-out gradient CPC method wasused to separate several CHAs from green coffee beans for analyticalpurposes (Romero-González & Verpoorte, 2009). However up to nowCPC was not used to purify green coffee extracts without salting-outto separate a whole group of chlorogenic acids.

When using CHAs as food additives a problem of their interactionwith amino acids, peptides and proteins may occur. This may cause aretardation of their absorption and a reduction of antioxidant activity,especially in foods submitted to heating (Rawel & Rohn, 2010). Thepresence of CHAs in food products may additionally result in a bittertaste. These compounds may furthermore undergo oxidative damage(both enzymatic and non-enzymatic), which may lead to a darkeningof products (Álvarez-Parilla et al., 2007; Budryn, Nebesny, Rachwał-Rosiak, & Oracz, 2013; Budryn & Rachwał-Rosiak, 2013). Because ofthese factors it may be beneficial to encapsulate chlorogenic acids byforming inclusion complexes with β-CD and to add them in this formto foods and food supplements (Nasirullah, Kumar, & Shariff, 2011;Szejtli & Szente, 2005). Previous studies by Paramera, Konteles, andKarathanos (2011) concerning the release of curcumin from thecomplex with β-cyclodextrin in simulated gastric and pancreatic fluidsshowed a high degree of release of polyphenolic compound from thecomplex by the digestive enzymes.

Previously described attempts of forming such complexes withCHAs regarded mostly pure 5-CQA (Chao, Wang, Zhao, Zhang, &Zhang, 2012; Álvarez-Parilla et al., 2010; Zhao, Wahg, Yang, & Tao,2010; Górnaś, Neunert, Baczyński, & Polewski, 2009. Some trials werealso made to form inclusion complexes of CHAs in roasted coffeeextracts to eliminate the bitter taste sensation (Hamilton & Heady,1970; Imamura et al., 1995).

The aim of thisworkwas to purify aqueous green coffee bean extractwith the use of a CPC technique to increase the concentration of CHAswith a simultaneous decrease of caffeine quantity. The second goalwas to obtain inclusion complexes of both, crude and purified extracts,with β-CD and to determine the influence of purification and com-plexation of extracts on their composition and antioxidant capacity.

2. Materials and methods

2.1. Chemicals and reagents

Analytical-grademethanol, ethanol and ethyl acetate were purchasedfrom Poch (Gliwice, Poland). HPLC-grade formic acid, acetonitrile andtriethylamine acetate (TEAA) were purchased from Fluka (St. Louis, MO,USA), trifluoroacetic acid (TFA) from Merck (Darmstadt, Germany),5-CQA (~98%), benzoic acid (BA, ~99%), caffeine (~99%), β-CD (~97%)and 2,2-diphenyl-1-picrylhydrazyl (DPPH, ~95%) from Sigma Aldrich(St. Louis, MO, USA), and nylon syringe filters from Chromacol (Herts,UK). Ultrapure water (resistivity, 18.2MΩ cm−1) was obtained from aMillipore Milli-Q Plus purification system (Bedford, MA, USA).

2.2. Preparation of crude green coffee extract (CGCE)

Green Robusta coffee beans (Coffea canephora L.), Conilon variety,harvested in Brazil in 2009, hulled by dry method, purchased fromBero Polska (Gdynia, Poland) with 8.3% water content were used.Beans were ground and sieved to a particle size ranging from 480 to680 μm. Very fine particles limiting the effectiveness of filtration, andrelatively large particles characterized by low extractability of phenoliccompounds were eliminated. The fraction after sieving comprisedapproximately 90% of the material obtained after grinding. The extractwas obtained using 400 g of ground coffee beans for one batch and1:5.75 (w/w) ratio of ground coffee towater. The suspensionwas boiledin a pressure vessel (PS-5682 Vienna, Austria) at 110 °C for 10 min,cooled in a water bath to a temperature of 40 °C for 20min and filtered

Please cite this article as: Budryn, G., et al., Inclusion complexes of β-cyclodextracts of green Robusta coffee ..., Food Research International (2013), htt

under vacuum using filter paper with basis weight of 84 g/m2 Poch(Gliwice, Poland) and a vacuum pump KNF 18 035.3 N (Neuberger, NJ,USA), according to Budryn et al. (2009). The extraction process wasrepeated three times and the extraction yield was 35.1%. The extractwas then frozen, freeze-dried in a DELTA 1-24LSC Christ freeze drier(Osterode am Harz, Germany) and stored at−25°C until analysis.

2.3. Purification of CGCE

To purify the extract CPC technique was used. A chromatographSPOT Prep II 50 from Armen Instrument (Saint-Avé, France) integratedwith a 2-channel UV/VIS detector and a fraction collector was used.CPC uses a two-phase liquid–liquid system without any solid phase.This method is based on the preparation of two phases – upper andlower – which are more and less hydrophobic, respectively. In mostcases two phases of three solvents are prepared. These phases areused as a stationary and mobile phases and can be shifted in relationto each other in a descending or an ascending mode. This techniqueuses the difference of partition coefficients of mixed componentsbetween two phases of a given solvent system (Kim et al., 2010).Initially a couple of various versions of two-phase systems wereprepared. To each of them a standard of 5-CQA was added and after16 h of equilibration partition coefficients (K) of 5-CQA between thetwo phases with the use of HPLC/DAD technique was established(Table 1), according to the equation: K = concentration of 5-CQA inupper phase/concentration of 5-CQA in lower phase. This procedureenabled us to choose a system that allowed receiving a partitioncoefficient of 5-CQA close to K = 1. This system was used to purifyCGCE. To its preparation water, ethanol and ethyl acetate were used ata ratio of 5:1:4 (v/v/v) (two-phase solvent system). CPC rotor was filledwith a hydrophilic stationary lower phase in 15min, with a flow rate of8mL/min and a speed 1400×g. Next 2.5 g of CGCE freeze-dried extractwas dissolved in 25 mL of a two-phase solvent system (10% of rotorvolume), purified on a nylon syringe filter 0.45 μm and injected onto arotor. Afterward a hydrophobic mobile upper phase was transferredthrough a rotor in an ascending mode with a flow rate of 30 mL/min,and a speed 450×g in 20min. During this period elution of componentwith amobile phase took place. After that a 15min extrusionwith lowerphase was done, in which the stationary phase containing retainedanalytes was entirely pumped out of the system preserving theirseparation pattern. Elution of CHAs occurred mostly from 20 to24 min and from 29 to 32 min of analysis. 25 mL volume fractionswere collected. Fig. 1 presents an exemplary chromatogram obtainedat a wavelength of 325 nm with Armen Glider CPC v5.0b.11 software.Fractions collected during time periods mentioned before, whichcontained CHAs, were combined and concentrated by the evaporationof ethanol and ethyl acetate using a ScanMaxiVac Labogene (Lynge,Denmark) concentrator. Parameters of the process were as follows:temperature 45 °C, pressure 120 mbar (2 h) and 75 mbar (3 h),



0

500

1000

1500

2000

2500

3000

3500

4000A

bsor

banc

e (m

AU

)

Time (min)

0 7 14 21 28 35

Fig. 1. Centrifugal partition chromatogram (CPC) of crude aqueous green coffee extract(CGCE);― 325 nm; - - - 280 nm.

3G. Budryn et al. / Food Research International xxx (2013) xxx–xxx

centrifugal speed 1550×g. The concentrate was then frozen and freeze-dried as in Section 2.2.

2.4. Chlorogenic acids and caffeine UHPLC/DAD quantitative analysis

The obtained crude and purified extracts were characterized interms of CHAs and caffeine concentrations. Solutions of extracts inultrapure water (20mg/mL) with an added internal standard (benzoicacid, 1mg/mL)were prepared. Then the solutions were filtered througha 0.20 μm nylon syringe filter and injected into UHPLC/DAD system.Chromatographic analysis was carried out using an UHPLC+ Ultimate3000 system equipped with an auto sampler and a DAD detector fromDionex (Sunnywale CA, USA), measuring the UV–VIS spectrum overa range of 190–900 nm. The analytical column Accucore™ C18(100 mm × 3.0 mm × 2.6 μm) from Thermo Scientific (Hudson, NewHamshire, USA) was used. The mobile phase consisted of water/formicacid mixture (99:1, v/v) (solvent A) and acetonitrile/water/formic acid(80:19.8:0.2, v/v/v) (solvent B)withflow rate of 0.5mL/min. The elutionwas performed in two steps: gradient from 5% B at 0 min to 35% B at23min, and then isocratic 35% B for 5 min. 2 μL injection volume and25 °C column temperature were used. Detection was performed attwo wavelengths, 280 nm for caffeine and 325 nm for chlorogenicacids, using Chromeleon 6.8.1 software. Caffeine was identified andquantified by comparing its retention time and UV spectrum withthose of reference standard (Budryn, Żyżelewicz, Nebesny, Oracz, &Krysiak, 2013). For CHA identification by LC–ESI-MS/MS analysis wasused. The quantification was performed using the internal standard(benzoic acid) and external standard (5-CQA) methods. All CHAswere quantified as 5-CQA equivalents. Calibration curves wereconstructed in the concentration range from 0.05 to 1 mg/mL ofstandards and were linear within the range. LOD and LOQ for 5-CQA were 0.028μg/mL and 0.089μg/mL, respectively and for caffeine0.022μg/mL and 0.070μg/mL respectively. Repeatability of analysis was97.9% for 5-CQA and 96.8% for caffeine.

2.5. Preparation of inclusion complexes of β-CD with 5-CQA,CGCE and PGCE

Inclusion complexes of β-CD with 5-CQA or β-CD with GCEs wereprepared by mixing those substances in an aqueous solution at atemperature of 50 °C in a Pierce Reacti-Therm TS-18821 reactor fromThermo Scientific (Hudson, New Hampshire, USA) for 2 h. Complexescontaining β-CD and 5-CQA or extracts were obtained using a molarratio 1:1 and 1:2. Therefore, 0.1135g of β-CD and 0.0354g of 5-CQA or0.1135 g of β-CD and 0.0708 g of 5-CQA were dissolved in 1 mL ofultrapure water. In the case of freeze-dried coffee extracts the same


amount of β-CD was mixed with 0.1094 g or 0.2187 g of CGCE, andalternatively 0.0651 g or 1.303 g of PGCE, which contained 0.0354 or0.0708g of CHAs, respectively calculated on 5-CQA. After complexationthe solution was left for 24 h at 0 °C and then the obtained suspensionwas centrifuged in MIKRO 22R centrifuge from Hettich (Kirchlengern,Germany) at 4°C for 20min at 10,000×g. The supernatant containingnon-complexed CHAs was collected and the precipitate wasadditionally extracted twice with 10 mL of methanol, the suspensionwas centrifuged, and the supernatant again collected. The threeobtained portions of supernatant were combined, concentrated undervacuum to a final volume of 3 mL, freeze-dried and subject to CHAquantitative analysis by UHPLC/DAD technique as explained inSection 2.4. Complexation efficacy was calculated using the formula:E= [(cintr− cfree) / ctheor] × 100%, cintr — concentration of CHAs in thesolution before complexation (g/mL), cfree — concentration of CHAsnon-bound in inclusion complexes and calculated on 5-CQA (g/mL),and ctheor — theoretical concentration of CHAs which form an inclusioncomplex with β-CD in a molar ratio of 1:1, calculated on 5-CQA,i.e. 0.0354g (g/mL).

2.6. Identification of CHAs and their complexes with β-CD with the use ofUHPLC/DAD-ESI-MS/MS and ESI-MS/MS techniques

The identification of CHAs in crude andpurifiedGCEswas performedby TurboFlow UHPLC/DAD-ESI-MS/MS method in a Transcend TLX-1chromatograph equipped with Q-Exactive tandem mass spectrometerfrom Thermo Scientific (Hudson, New Hampshire, USA). The UHPLCsystem consisted of purifying TurboFlow cyclone-P 50 × 0.5m columnand analytical Hypersil GOLD 50 × 2.1 mm, 1.9 μm column, both fromThermo Scientific (Hudson, New Hampshire, USA), each supplied witha loading or analytical pump respectively. The TurboFlow UHPLCmethod consisted of three steps: 1. extraction step — the TurboFlowcolumn removes a significant portion of the large molecules present inthe extract; 2. transfer step — elution of the analytes from theTurboFlow column to an analytical column; 3. gradient elution of theanalytes on an analytical column. 10 μL of 0.1mg/mL GCEs solution ofextracts in ultrapure water was injected into the Turboflow column.The operating temperature was maintained at 30 °C. The HPLC mobilephase consisted of eluent A, a 50 μM aqueous solution of TEAA andeluent B, a 50 μM solution of TEAA in 10:90, water: acetonitrile (v/v).The elution program was as follows:

exp:

Time (s)

trinwith//dx.doi.o

Loading pump

chlorogenic acids (CHArg/10.1016/j.foodres.20

s) from cr13.10.013

Analytical pump

Eluent (%)
Flow rate(mL/min)
Eluent (%)

ude and pu

Flow rate(mL/min)

A
B A B
rified a

0–15
100 0 1.50 Isocratic 100 0 0.80 Isocratic 15–30 100 0 0.13 Isocratic 100 0 0.80 Isocratic 30–90 100 0 0.13 Isocratic 100 0 0.67 Isocratic 90–150 60 40 1.50 Isocratic 90 10 0.80 Gradient 150–270 0 100 1.50 Isocratic 90 10 0.80 Isocratic 270–315 100 0 1.50 Isocratic 100 0 0.80 Gradient
The chromatogram was recorded at 325 nm with the use of a DADdetector from Dionex (Sunnywale CA, USA) measuring the UV–VISspectrum over a range of 190–900 nm and at Q-Exactive hybridquadrupole-Orbitrap mass spectrometer using Aria 1.6.3, ThermoXcalibur 2.2. and Qexactive Tune 2.1 software, respectively. Datawere acquired in full scan MS and target MS2 scanning mode to detectspecific mass ions of CHAs. For caffeoylquinic acids (CQAs) m/z 353,for feruloylquinic acids (FQAs) m/z 367 and for dicaffeoylquinicacids (diCQAs) m/z 515 were used (Clifford, Kirkpatrick, Kuhnert,Roozendaal, & Rodrigues Salgado, 2008). Capillary temperature wasset at 320 °C, and the nebulizer and collision gas was nitrogen. Thecollision energy was 35 eV and ionization voltage was 3.0 kV. The fullscan mass spectra of the investigated compounds were recorded in

queous


Table 2Chlorogenic acids and caffeine concentrations in crude (CGCE) and CPC purified (PGCE)green coffee extracts (g/100 g db.).

Compound CGCE PGCE

3-O-Caffeoylquinic acid (3-CQA) 6.06±0.57 11.02±0.855-O-Caffeoylquinic acid (5-CQA) 9.18±0.78 16.82±1.064-O-Caffeoylquinic acid (4-CQA) 7.30± f0.61 13.28±0.873-O-Feruloylquinic acid (3-FQA) 1.79±0.11 2.38± 0.175-O-Feruloylquinic acid (5-FQA) 1.11±0.09 1.98± 0.074-O-Feruloylquinic acid (4-FQA) 2.20±0.17 3.79± 0.233,4-di-O-Caffeoylquinic acid (3,4-diCQA) 1.91±0.08 2.51± 0.123,5-di-O-Caffeoylquinic acid (3,5-diCQA) 1.23±0.05 0.83± 0.044,5-di-O-Caffeoylquinic acid (3,5-diCQA) 1.59±0.08a 1.73± 0.09a

Total chlorogenic acids 32.37 54.35Caffeine 5.17±0.46 nd

nd—not detected (LOD≤ 0.022 μg/mL); ±standard deviation; n = 6; different letters orthe lack of them in a row corresponds to significant differences (p b 0.05).


negative ionizationmode over the rangem/z 100–2000. Identification ofmajor CHAs was performed by comparison of the molecular weight offragment ions, and positional isomers were specified on the basis oftheir gradient polarity, hence the order of elution, and the proposedorder is consistent with the great majority of studies, for example byFarah, de Paulis, Trugo, and Martin (2005). The confirmation of formedinclusion complexes was performed by negative ESI-MS/MS mode,based on Dotsikas and Loukas (2003), with some modifications.Experiments were performed on Q Exactive hybrid quadrupole-Orbitrap mass spectrometer. Inclusion complexes were dissolved in a50 μM TEAA aqueous solution (10 μg/mL) and directly infused intoelectrospray ion source (ESI) via syringe pump at a flow rate of5 μL/min. The capillary temperature was 320 °C, nebulizer gas andcollision gas was nitrogen, and the source voltage was 3.0 kV. Thecollision energy was 18 eV. The complexes were analyzed using fullscan MS and target MS2 in negative ionization mode with a scan rangefrom 100 to 2700m/z. Peaks with a negatively charged molecular ion([M –H]−) at m/z 1488, 1502 and 1651 were identified, respectively,as β-CD-CQAs, β-CD-FQAs and β-CD-diCQAs inclusion complexes.

2.7. Thermodynamic analysis

The extent of changes of β-CD inclusion complexes with CHAs uponheating was estimated based on thermal profile evaluated by the DSCtechnique (Zhao et al., 2010). The method allows the characterizationof phase transitions or chemical interactions, i.e. temperature andenthalpy by measuring the quantity of emitted or absorbed enthalpyduring heating or cooling of the sample. Thermal analysis was con-ducted using a DSC 111 Setaram Instrumentation (Caluire, France).Heating of inclusion complexes or GCEs, 5-CQA, β-CD or mixture ofthe last two (60 mg in a sealed hermetically stainless steel pan) wascarried out in the temperature range of 25–200 °C. Heating rate was2 °C/min. Results of the thermal analysis were processed using thescanning calorimeter software. Melting or other transition tempera-tures were determined based on peaks of thermal profile curves andthe enthalpies of transition were calculated based on the surface areasunder peaks. Calibration of temperature and energy was carried outusing an indium standard at the same scanning rate as for testedsamples.

2.8. Antioxidant activity of chlorogenic acids and β-CD-CHA complexes

2.8.1. Redox potentialRedox potential analysis was carried out using aqueous solutions

of 5-CQA, β-CD GCEs or derived thereof inclusion complexes at con-centrations of 2 mg/mL. Redox potential of 25 mL of a solution wasmeasured using a platinum BlueLine 31 RX redox electrode and a SchottCG 843 voltmeter (Schott, Mainz, Germany) according to Nicoli,Toniolo, and Anese (2004). Calibration was performed against a redoxstandard solution (Reagecon Diagnostics Ltd., Shannon, Ireland). Priorto analysis oxygen was removed from the system by continuousnitrogen flushing for 10 min and the measurement was conducted innitrogen atmosphere. The redox potential was monitored for at least5min at 25 °C, until an arbitrary stable result was achieved, defined asthat changing less than 2mV in a 3min period.

2.8.2. Scavenging capacity against DPPH• radicalScavenging capacity against DPPH• radical was determined as

described by Scherer and Godoy (2009) with some modifications.DPPH• was used as a standard radical. The test was carried out usingaqueous solution of 5-CQA, β-CD, GCEs or inclusion complexes derivedtherefrom at concentrations of 0.5, 1.0, 1.5 and 2.0 mg/mL of eachsubstance. 0.1mL of a solution was reacted with 3.9mL of methanolicradical solution (DPPH•:methanol, 1:20000 (w/w). Methanol was usedas a blank sample and 0.1 mL of water with 3.9 mL of methanolicDPPH• solution as a control. Based on the measurement of absorbance


of a tested sample (Atest) and control (Acontrol) after 30min reaction inthe darkness at 517 nm (UV/VIS spectrophotometer U-2800 A Hitachi(Tokyo, Japan) a calibration curve of concentration of a sample versusscavenging capacity against DPPH• radical and a regression equationwere obtained. Scavenging capacity against DPPH• radical equaled:%=[(Acontrol–Atest)/Acontrol]×100%. Then the concentration of a sampleatwhich the quantity of the radical formwas reduced by 50% (IC50) wascalculated from the regression equation.

2.9. Statistical analysis

The extracts were prepared twice and each was subjected tocomplexation. Analyses were carried out in triplicate and their resultswere subjected to statistical analysis. It comprised determination ofaverage values of six measurements and their standard deviation aswell as one-way ANOVA (analysis of variation) using Statistica 10.0software at the significance level pb0.05.

3. Results and discussion

3.1. Selections of the two-phase solvent system for CGCE purification

CGCE contained 32.37 g of CHAs (Table 2). Other compounds werecaffeine, carbohydrates, proteins, minerals, and soluble fiber (Budryn& Nebesny, 2012). Firstly a two-phase solvent system was selected toseparate chlorogenic acids from other compounds in CPC. This type ofseparation allows the use of high sample loading capacities comparedto techniques with solid support. It enables 100% sample recovery, lesssolvent usage, cost saving and finally greater tolerance of substancesthat can damage solid filling of a column. Various solvent systemswith different volume ratios were tested (Table 1). In most systemswater with 1% of TFAwas used to increase a gradient of system polarity,however it caused an excessive dissolution of 5-CQA in the upper, morehydrophobic layer (KN 1), especially in the system of water+1% TFA–ethanol–ethyl acetate. Ultimately acidification was abandoned and thesystem of water–ethanol–ethyl acetate at a ratio of 5:1:4 (v/v/v) withK = 1.17 for 5-CQA was chosen. The same system was proposed byKim et al. (2010), who obtained K=0.83 for 5-CQA from blackcurrantleaves, which could indicate that in those studies 5-CQA was dissolvedbetter in the lower phase. Fig. 1 shows the preparative CPC separationchromatogram of green coffee aqueous fraction. The chromatogramwas collected at wavelengths of 325 and 280 nm in order to visualizefractions containing CHAs and caffeine, which absorb UV at bothwavelengths, however CHAs have maximal absorption at 325 nm andcaffeine at 280 nm. According to this, fractions collected between20 and 24 min (diCQAs and 4-&5-FQAs) and those between 29 and32min (CQAs and 3-FQAs)were chosen as those containing chlorogenicacids. The latter collected fraction showed a similar absorbance at both



Fig. 2.UHPLC chromatogram of lyophilized (A) crude and (B) purified green coffee extract, recorded using UV detection at 325nm. Peak designations: 1. 3-CQA; 2. 5-CQA; 3. 4-CQA; 4. 3-FQA; 5. caffeine; 6. 5-FQA;7. 4-FQA; 8. 3,4-diCQA; 9. 3,5-diCQA; 10. 4,5-diCQA; for chlorogenic acid abbreviations see Table 2.


325 and 280nm, howeverHPLC analysis revealed that it contained CHAsbut not caffeine (Fig. 2). The chromatogram indicates different partitioncoefficients of particular CHAs. In those conditions a part of the CHAswas eluted during the change of elution phase to extrusion phase(compounds with K ~ 1, 20–24 min of separation), while a significantpart also during second phase of extrusion (compounds with K b b1,29–32min of separation).

3.2. Influence of CPC purification of CGCE on CHAs composition

One step purification of aqueous CGCE allowed the increase CHAconcentration from 32.37 to 54.35 g 100 g db and the elimination ofcaffeine (Table 2, Fig. 2). Kim et al. (2010) purified an extract ofblueberry leaves and obtained a 96% 5-CQA preparation. However theextract of crude blueberry leaves contained only a few compounds,and among CHAs practically only 5-CQA, which was eluted as a sharppeak in the short period of the analysis. CGCE was a complex mixtureand individual CHAs eluted in various retention times. Collecting widefraction of CHAs resulted in its much greater impurity. Identification ofCHAs was performed by the UHPLC/ESI-MS/MS technique (Clifford,


Johnston, Knight, & Kuhnert, 2003; Perrone, Farah, Donangelo, dePaulis, & Martin, 2008). The first three peaks, isomers of CQAs, werecharacterized by negative mode with MS precursor ions m/z 353 andproduced MS2 fragment ions at m/z 179, consistent with the presenceof caffeic acid and residue. The next three peaks, isomers of FQAs,were characterized with MS precursor ions m/z 367 and producedMS2 also fragment ions at m/z 193, consistent with the presence of aferulic acid residue. The last three peaks, isomers of diCQAs, werecharacterized with MS precursor ions m/z 515 and produced MS2

fragment ions atm/z 353, consistent with the presence of a CQA residue(Clifford, Knight, Surugu, & Kuhnert, 2006). CGCE composition indicatedon a partially intramolecular transacylation of CHAs during aqueousextraction (Budryn et al., 2009; Farah, de Paulis, Moreira, Trugo, &Martin, 2006). CHAs in green coffee beans consist mostly of 5-CQA,which concentration is about 60%, while 3-&4-CQA amount to about10% of all chlorogenic acids. High temperature, especially in aqueoussolutions causes a decrease of 5-CQA to about 30% and an increase of3-&4-CQA to 25% each. In case of CGCE, 5-CQA content amounted 28%of total CHAs, 3-CQA - 18% and 4-CQA - 23% that indicates to a partialtransacilation of 5-CQA during extraction. During CPC purification




CHAs composition did not change drastically. 5-CQA concentrationamounted 31%, while 3-CQA and 4-CQA 20 and 24%, respectively. It isworth mentioning that industrial elimination of caffeine also causesCQA transacacylation, and even to a greater extent (Farah et al., 2006).Overall the concentration of CQAs increased due to purification from70 to 76%. This happened at the expense of diCQAs, whose contentdecreased from 15 to 9%. FQAs remained on the same level of 15%.DiCQAs were probably partially eluted between 20 and 24 min ofseparation, however due to the presence of caffeine (Fig. 1) thesefractions were not collected. To summarize as a result of CGCE

Fig. 3.MSspectra of inclusion complexes ofβ-CDwith (A) CQAs;m/z 1488, (C) FQAs;m/z 1502 acomplexes of β-CD with (B) CQAs; m/z 353, (D) FQAs;m/z 367 and (F) diCQAs; m/z 515.


purification by the CPC technique, a PGCE with over a 1.5-fold higherconcentration of CHAs and free of caffeine was obtained. The onlychemical reagent harmful to health used in the purification processwas ethyl acetate, which can be easily eliminated from PGCE duringits concentration. Ethyl acetate belongs to “class 3 solvents”, towhich daily exposure of 50 mg per day is allowed and may beregarded as less toxic and with lower risk to human health (FederalRegister, 1997). Because of this, the purified preparation is safeand just like CGCE can be used to enrich food products and foodsupplements.

nd (E) diCQAs;m/z 1561, andMS2 spectra of fragment ions after fragmentation of inclusion



Fig. 3 (continued).


3.3. Identification of β-CD inclusion complexes with CHAs from GCEs andthe efficacy of their formation

Earlier studies of other authors were performed to evaluate thecomplexation procedure, namely to determine the binding constant,and the stoichiometry of complexes of 5-CQA with β-CD. Usingtechniques such as 1H NMR, ITC (isothermal titration calorymetry),spectrophotometry and fluorimetry they proved that 5-CQA forms aninclusion complex with β-CD at a molar ratio of 1:1 (Álvarez-Parillaet al., 2010; Chao et al., 2012; Zhao et al., 2010). LC–MS studies causesome limitations because of complex instability of organic solventsas well as under increased pressure. Electrospray ionization massspectrometry (ESI-MS) provides a powerful tool to study non-covalent


“host–guest” inclusion complexes of a CD and hydrophobic group ormolecules. The mild ionization procedure allows them to maintaintheir complex structure (Dotsikas & Loukas, 2003). Identificationof inclusion complexes of β-CD with CHAs was performed withthe use of ESI-MS/MS technique. Complexes of β-CD with CQAs werecharacterized with MS precursor ions m/z 1488 and produced MS2

fragment ions atm/z 353, consistent with the presence of a CQA residue(Fig. 3). Complexes of β-CD with FQAs were characterized with MSprecursor ions m/z 1502 and produced MS2 fragment ions at m/z 367,consistentwith the presence of FQA residues. Existence of such complexwas proved for the first time. In their earlier studies Górnaś et al. (2009)concluded that ferulic acid, and therefore also FQA, is unable to createinclusion complexes with β-CD due to a presence of methylated



Fig. 3 (continued).


hydroxyl group in its structure. However, formation of similarcomplexes was observed by Paramera et al. (2011), who identified acomplex of β-CD with curcumin, which contains two molecules offerulic acid in its structure. Complexes of β-CD with diCQAs, werecharacterized with MS precursor ions m/z 1651 and produced MS2

fragment ions at m/z 515, consistent with the presence of a diCQAresidue. Recorded ion molecular masses confirmed a molecular ratio1:1 ofβ-CDandCHAs in inclusion complexes. No specific ions consistentwith the presence of β-CD in a molar ratio 2:1 with CHAs, especiallydiCQAs were detected. Proposed structure of created complexes isshown in Fig. 4, where the phenolic group is included inside theβ-cyclodextrin cavity (Górnaś et al., 2009).


Fig. 5 shows the DSC thermograms of 5-CQA, β-CD, their physicalmixture and inclusion complex, as well as complexes of β-CD withCHAs from CGCE or PGCE. The DSC thermal profiles of β-CD and 5-CQA showed the endothermic peaks at 143 and 199 °C respectively,which are similar to results obtained by Zhao et al. (2010). The firstpeak was associated with the melting point of 5-CQA and the secondwith elimination of water molecules from the β-CD cavity (Parameraet al., 2011). The DSC thermal profile of 5-CQA and β-CD physicalmixture prepared in 1:1molecular ratio showed twowide endothermicpeaks, obtained due to changes mentioned above. However they werecharacterized by not as steep transition temperatures as in the case ofheating individual substances, probably due to electrostatic interactions



Fig. 4. Proposed chemical structures of β-CD inclusion complexes with (a) 3-CQA, (b) 3-FQA and (c) 3,5-diCQA.


and adduct formation. The inclusion complex β-CD-5-CQA showed adouble endothermic peak at 173 and 178 °C, completely different fromthose for 5-CQA and β-CD mixture. Two local minima of temperatureof an endothermic transition of the complex may be linked to de-composition of its two forms (Górnaś et al., 2009). This thermic profile

Temperature/ °C

140 150 160 170 180 190

Heat Flow/ mWExo

5-CQA

β-CD

β-CD-5-CQA complex

β-CD-CGCE complex

β-CD-PGCE complex

200

β-CD+5-CQA

Fig. 5. Differential scanning calorimetry (DSC) thermograms of β-cyclodextrin (β-CD), 5-caffeoylquinic acid (5-CQA), their physical mixture (β-CD + 5-CQA) and inclusioncomplex (β-CD-5-CQA complex), and complexes of β-cyclodextrin with chlorogenicacids from green coffee extracts: crude (β-CD-CGCE complex) and purified (β-CD-PGCEcomplex).


confirms inclusion complex formation, since disappearance of thermaleffect of the included 5-CQA molecule is considered as a proof ofinclusion instead of adduct formation. β-CD complexes with CHAsfrom CGCE and PGCE showed different thermal profiles than with of5-CQA, with wide peaks at 155 and 160 °C, respectively. This indicatesa variety of obtained complexes and therefore included not only 5-CQA but also other CHAs, such as FQAs and diCQAs.

Efficiency of CHA complexation amounted from 18 to 38%. Thatcorresponds to 19 - 76% of a maximal amount of chlorogenic acids,which could be bound in an inclusion complex with β-CD in a molarratio 1:1 (Table 3) (Nasirullah et al., 2011; Paramera et al., 2011; Zhaoet al., 2010). In our studies the complexation efficiency increased as aresult of extract purification and also of increase from 1:1 to 1:2 molarratio of β-CD to CHAs from GCEs during complexation. Regardless ofthe extracts' purity and molar ratio of CHAs to β-CD, the highestefficiency of complexation was observed for diCQAs, which amountedon average from 34 to 55%. Lower efficiency was observed for FQAs —on average from 27 to 42%. The lowest complexation efficiency, onaverage from14 to 34%, showed inCQAs,whichwere present in extractsat the highest concentration. This indicates that less polar moleculeswere more susceptible to inclusion into hydrophobic β-CD cavity.Among CQAs the highest complexation efficiency was observed in the5- isomer, in the case of FQAs in the 4- isomer, and among diCQAs inthe 4,5-isomer. Efficiency of β-CD complexation with a 5-CQA standardamounted to 26 and 40% for molar ratios of 1:1 and 1:2 in a complexingmixture respectively, therefore it was on a similar level with com-plexation efficiency of this compound from PGCE, which amountedto 26 and 36%, respectively for molar ratios of 1:1 and 1:2. The highest5-CQA concentration in complexes with β-CD amounted to 20%.According to this supplementing of food product or food supplementwith therapeutically required amount of CHAs implies introduction of4-fold higher amount of β-CD. In case of PGCE the concentration ofCHAs is much higher and amounts to about 50% and the other 50% areproteins, carbohydrates, minerals and soluble fiber. However β-CD



Table 3Efficacy of formation of β-cyclodextrin (β-CD) and chlorogenic acid inclusion complexes using crude (CGCE) and purified (PGCE) green coffee extracts.

Chlorogenic acid Efficiency of complexation (%)

β-CD:CGCE 1:1a β-CD:PGCE 1:1a β-CD:CGCE 1:2a β-CD:PGCE 1:2a

3-CQA 14.60± 1.12 18.78± 1.24a 20.17±1.54a 32.74± 2.135-CQA 18.36± 1.38 26.40± 1.55a 29.61±1.78a 35.82± 2.564-CQA 8.47±0.65 14.96± 0.82 20.55±0.83 33.83± 2.933-FQA 21.27± 1.76a 25.71± 1.38b 23.15±1.71a,b 33.04± 2.435-FQA 30.06± 2.08a 33.47± 2.17a 33.55±2.17a 43.11± 3.114-FQA 30.03± 1.87 46.18± 3.39a 46.11±3.62a 48.95± 2.97a

3,4-diCQA 29.08± 1.69 40.01± 2.77a 40.88±2.17a 59.87± 3.583,5-diCQA 30.85± 1.91 45.58± 3.46a 38.75±2.55 48.72± 3.74a

4,5-diCQA 40.58± 2.97a 47.42± 2.63b 45.36±2.71a,b 56.29± 4.17Total chlorogenic acids 18.98 25.25 28.75 37.80

Mass ratio β-CD:chlorogenic acids in inclusion complexes100:5.92 100:7.88 100:17.93 100:23.58Molar ratio β-CD:chlorogenic acids in inclusion complexesa

1:0.19 1:0.25 1:0.57 1:0.76Content of chlorogenic acids in inclusion complexes (g/100 g)5.59 7.30 15.20 19.08Efficacy of complexation of β-CD with 5-CQA standard (%)β-CD:5-CQA standard 1:1a β-CD:5-CQA standard 1:2a

26.15± 1.87 40.38±2.74Mass ratio β-CD:5-CQA standard in inclusion complexes100:8.16 100:25.19Molar ratio β-CD:5-CQA standard in inclusion complexes1:0.26 1:0.81Content of 5-CQA standard in inclusion complexes (g/100 g)7.54 20.12

3-CQA, 3-O-caffeoylquinic acid; 5-CQA, 5-O-caffeoylquinic acid; 4-CQA, 4-O-caffeoylquinic acid; 3-FQA, 3-O-feruloylquinic acid; 5-FQA, 5-O-feruloylquinic acid; 4-FQA,4-O-feruloylquinic acid; 3,4-diCQA, 3,4-O-dicaffeoylquinic acid; 3,5-diCQA, 3,5-O-dicaffeoylquinic acid; 4,5-diCQA, 4,5-O-dicaffeoylquinic acid;

a Molar ratio calculated on 5-CQA in a mixture prepared for complexation; ±standard deviation; n= 6; different letters or the lack of it in a row corresponds tosignificant differences (p b 0.05).


complex has benefits like its sweet taste and low digestive absorption(in relation to β-CD), thus it can partly replace saccharose, simul-taneously decreasing the calorific value of a product (Szejtli & Szente,2005). Taking into account the research on curcumin by Parameraet al. (2011), it can be assumed that CHAs after breakdown of thecomplexes by digestive enzymes become fully bioavailable, but thisshould be confirmed in similar studies on CHAs.

3.4. Antioxidant capacity of inclusion complexes of β-CD with CHAs

Antioxidant capacities of individual substrates and formed com-plexes were studied with the use of redox potential measurement andscavenging capacity against DPPH• radical (Table 4). Solution of β-CDshowed very low antioxidant capacity measured by redox potential,i.e. 335.3 mV, because distilled water showed redox potential of340.1mV. Solution of CGCE showed much higher antioxidant capacity,namely 220.5mV and CPCpurification caused an increase of antioxidant

Table 4Antioxidant capacity of crude (CGCE) and CPC purified (PGCE) green coffee extracts andtheir complexes with β-cyclodextrin (β-CD).

Substance Redox potential(mV)

Scavenging capacityagainst DPPH• radical IC50(mg μmol−1 DPPH•)

β-CD 335.3± 24.1f 25.82±1.765-CQA 193.1± 11.3a 0.87± 0.05CGCE 220.5± 13.8b 1.47± 0.08PGCE 203.4± 9.5a,b 1.10± 0.04β-CD-5-CQA 1:1a 292.6± 10.8 3.32± 0.21c

β-CD-5-CQA 1:2a 254.3± 11.8c 2.86± 0.18a

β-CD-CGCE 1:1a 308.1± 17.6e,f 4.91± 0.35β-CD-CGCE 1:2a 301.2± 15.2e 4.14± 0.27β-CD-PGCE 1:1a 287.5± 18.1d,e 3.15± 0.14b,c

β-CD-PGCE 1:2a 277.6± 13.5c,d 3.06± 0.23a,b

a Molar ratio calculated on 5-CQA in mixture prepared for complexation; 5-CQA, 5-O-caffeoylquinic acid; ±standard deviation; n = 6; different letters or the lack of it in acolumn corresponds to significant differences (p b 0.05).


capacity (p b 0.05) to 203 mV. It resulted from the increasedconcentration of CHAs in PGCE comparing to CGCE. Redox potentialof 5-CQA was 193.4 mV, so both extracts showed lower antioxidantcapacity than the pure standard. Similar tendency was observed bymeasurement of scavenging capacity against DPPH• radical, whereIC50 of β-CD amounted 25.8mg/μmol DPPH• and for crude and purifiedGCE it was 1.47 and 1.10mg/μmol DPPH•, respectively. For 5-CQA thiscapacity was 0.87mg/μmol DPPH•, so again higher than for GCEs.

Inclusion complexes of β-CD and CHAs from GCEs showed lowerantioxidant capacity than CGCE and PGCE. The same applied to thestandard 5-CQA and gained from it the inclusion complex. In the caseof complexes obtained from 1:2 molar ratio of β-CD to CHAs fromGCEs, CGCE showed a 2.5-fold, and PGCE 1.6-fold higher redox potentialthan the formed inclusion complex. Scavenging capacity against DPPH•radical was for CGCE and PGCE, respectively 2.3-fold and 2.6-fold higherthan the corresponding mixtures of derived inclusion complexes.However observed tendency may result mostly from the differenceof CHA concentration in GCEs and in inclusion complexes. The con-centration of CHAs in CGCE amounted 32.37 g/100 g, while in β-CD-CGCE complex it amounted to 15.20 g/100g, so was 2.13 times than inβ-CD-CGCE complex, and in PGCE the concentration of CHAs amountedto 54.35g/100g, while in β-CD-PGCE complex it was 19.08g/100g, andtherefore was 2.85 times than in β-CD-PGCE complex. Hence it couldbe observed that the antioxidant capacities of CHAs in complexeswere even higher than in initial extracts. In relation to the standard of5-CQA a similar tendency was observed. High antioxidant capacity of5-CQA after its inclusion to β-CD was also observed by Chao et al.(2012), Zhao et al. (2010) and Álvarez-Parilla, de la Rosa, Torres-Rivas,Rodrigo-Garcia, and González-Aguilar (2005). Mercader-Ros, Lucas-Abellán, Fortea, Gabaldón, and Núñez-Delicado (2010) observed thesame phenomena for various flavonols, and Lu, Cheng, Hu, Zhang, andZou (2009) for resveratrol. However, Álvarez-Parilla et al. (2007) andFayad, Marchal, Billaud, and Nicolas (1997) proved an effect of alimitation of enzymatic oxidation of CHA as a result of formation of aninclusion complex with β-CD and Szejtli and Szente (2005) pointed to




masking the bitter taste of 5-CQA as a result of formation of a complexwith β-CD. Therefore on the one hand, bound CHAs react with freeradicals, and on the other hand, they do not react with catecholasesand do not interact with the proteins of the mouth cavity (bitter testsensation). These slightly contradictory results regarding the reactivityof phenolic group included in a complex still need further research toexplain the mechanism of maintaining high antioxidant capacity evenafter inclusion in a complex. Previous studies supply probable solutionsto this issue, for example the stabilization of phenolic radical form in theCD cavity (Jullian et al., 2008) or the formation of intermolecularhydrogen bonds between the host and the guest (Chao et al., 2012).Better protection against oxidation of CHAs in inclusion complexeswith β-CD causes their higher stability and lack of darkening (Gacche,Zore, & Ghole, 2003), while they still remain highly bioavailable andantioxidant active (Mantegna et al., 2012; Paramera et al., 2011). Theantioxidant capacity of inclusion complexes should be furtherconfirmed in assays with biological radicals to prove their chainbreaking ability in food or in vivo.

4. Conclusions

In the present work aqueous crude green coffee extract was purifiedby CPC. As a result of purification the concentration of CHAs in theextract was increased by about 50%. The purification ofthe extractincreased the complexation efficiency of CHAs with β-CD, as well as ahigher molar ratio of CHAs from CGEs to β-CD in a complexationmixture. Complexation was proved for CQAs as well as for FQAs anddiCQAs, which in the case of the latter two was done for the first time.Antioxidant capacity of formed inclusion complexes was lower thanthat of corresponding extracts. However taking into account only theconcentration of CHAs in extracts and derived complexes, an increaseof CHAantioxidant capacity can be observed as a result of complexation.Obtained complexes can be used to enrich food products and foodsupplements in bioactive chlorogenic acids, which in the form ofinclusion complexes are more stable, their darkening and causing abitter taste limited and additionally probably retaining their highbioavailability.

Acknowledgments

The authors are grateful for the financial support provided by theNational Center of Science (project no. UMO-2011/03/B/NZ9/00745).

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Inclusion complexes of β-cyclodextrin with chlorogenic acids (CHAs) from crude and purified aqueous extracts of green Robusta coffee beans (Coffea canephora L.)