Influence of Coffee Genotype on Bioactive Compounds and the in

Article pubs.acs.org/JAFC

Influence of Coffee Genotype on Bioactive Compounds and the in Vitro Capacity To Scavenge Reactive Oxygen and Nitrogen Species Naira Poerner Rodrigues,† Terezinha de Jesus Garcia Salva,§ and Neura Bragagnolo*,† †

Faculty of Food Engineering, University of Campinas (UNICAMP), 13083-862 Campinas, São Paulo, Brazil Coffee Center, Agronomic Institute of Campinas (IAC), P.O. Box, 28, 13001-970 Campinas, São Paulo, Brazil

§

S Supporting Information *

ABSTRACT: The influence of green coffee genotype on the bioactive compounds and the in vitro antioxidant capacity against the principal reactive oxygen (ROO•, H2O2, HO•, and HOCl) and nitrogen (NO• and ONOO−) species of biological relevance was investigated. This is the first report on the capacity of green coffee to scavenge H2O2, HOCl, and NO•. Variations in the contents of total chlorogenic acids (22.9−37.9 g/100 g), cinnamoyl−amino acid conjugates (0.03−1.12 g/100 g), trigonelline (3.1−6.7 g/100 g), and caffeine (3.9−11.8 g/100 g) were found. Hydrophilic extracts of Coffea canephora and Coffea kapakata were the most potent scavengers of ROO•, H2O2, HO•, NO•, and ONOO− due to their chlorogenic acid contents, which were, on average, 30% higher than those found in Coffea arabica and Coffea racemosa. The results showed that genotype is a determinant characteristic in the bioactive compound contents and consequently in the antioxidant capacity of green coffee. KEYWORDS: Rubiaceae, cultivars, varieties, germplasm bank, reducing capacity, phenolic compounds, methylxanthines

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established.16 Oxidative and nitrosative stress, resulting from a disequilibrium between the antioxidant defense of the cells and the generation of oxygen (ROS) and nitrogen (RNS) reactive species, causes damage to some important cell components, including lipids, proteins, and DNA. Due to these effects, oxidative and nitrosative stress can cause human diseases such as heart and degenerative diseases, diabetes, cancer, and nearly all liver pathologies and also take part in the aging process.17,18 Antioxidants consumed in the diet are important to maintain the equilibrium of ROS and RNS, especially when the endogenous antioxidant defense system is not capable of efficiently deactivating the reactive species generated during oxidative and nitrosative stress.18 Several epidemiological studies indicated that the consumption of green coffee extracts had an antihypertensive effect in rats and humans,19 improved vasoreactivity in humans,20 had an inhibitory effect on the accumulation of fat and on the increase in body weight in rats and humans,21,22 and modulated glucose metabolism in humans.9 Health effects associated with coffee consumption largely depend on the bioacessibility and bioavailability of its bioactive compounds. After coffee ingestion, chlorogenic acids appear in the circulatory system as methylated, sulfated, and glucuronated metabolites in plasma concentrations that rarely exceeds nanomolar. Substantial quantities of both the parent compounds and their metabolites pass to the colon, where they are degraded by the action of the local microbiota, giving rise principally to small phenolic acid and aromatic catabolites that are absorbed into the circulatory system.23 With regard to caffeine bioavailability, rapid absorption, metabolization, and

INTRODUCTION Coffee plants belong to the Rubiaceae family and to the genus Coffea, which has 103 species. Coffea arabica (arabica coffee) and Coffea canephora (robusta coffee) are the only species used for commercial production, representing about 60 and 40%, respectively, of the world coffee market.1,2 The other coffee species, although having no commercial relevance, are important for genetic improvement of coffee plants, because they constitute gene reserves for resistance against pests, diseases, and adverse environmental conditions. Coffee species show considerable variation in their morphological, agronomical, and biochemical characteristics.3 The biochemical differences are responsible for the variations in chemical composition of the coffee fruits and even between genotypes of the same coffee species.3,4 Green coffee beans are rich in bioactive compounds, with a highlight on caffeine, trigonelline, chlorogenic acids,5 tocopherols (α, β, γ), and diterpenes (mainly kawheol and cafestol).6 A number of health properties were reported for these specific compounds in the literature. Caffeine presents various biological activities such as stimulation of the central nervous system, diuretic effect, peripheral vasoconstriction, smooth muscle relaxation, and myocardial stimulation.7 Trigonelline exerts antidiabetic action by decreasing serum glucose and increasing the insulin sensitivity index.8 Chlorogenic acids have been related to a series of health benefits such as reduction in type 2 diabetes9 and antihypertensive activity,10 as well as antibacterial, anti-inflammatory11,12 and antioxidant13 activities. Vitamin E, represented by the tocotrienols and the tocopherols, is a potent antioxidant, anti-inflammatory, and anticarcinogenic agent.14 Diterpenes present anticarcinogenic properties and stimulate the intracellular antioxidant defense mechanisms.15 The inverse relationship between the ingestion of foods rich in antioxidants and the decreased risk for the development of some chronic-degenerative diseases has already been well © XXXX American Chemical Society

Received: January 28, 2015 Revised: April 23, 2015 Accepted: April 24, 2015

A

DOI: 10.1021/acs.jafc.5b00530 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

genotypes are commercially cultivated (3 C. arabica and 5 C. canephora of the varieties Robusta and Kouilou). All of the plants are maintained in the Coffee Germplasm Bank and are cultivated in the experimental center of the Campinas Agronomy Institute (IAC) in Campinas, SP, Brazil, located at a latitude of 22°54′ S, a longitude of 47°05′ W, and an altitude of 640 m. Coffee fruits were harvested in the cherry maturation stage during the 2010/ 2011 harvest. Sample Preparation. After harvesting, hulling, and manual removal of the mucilage, the coffee endosperms with their parchment were immediately frozen in liquid nitrogen. The parchment and silver skin were manually removed; the endosperms were frozen in liquid nitrogen and freeze-dried for 129 h at −65 °C below 40 μm Hg (Liobras, São Paulo, Brazil). The freeze-dried endosperms were ground, sieved through a 0.5 mm sieve (ABNT 35, Tyler 32), vacuum packed (Jumbo Plus, Selovac, São Paulo, Brazil) in polyethylene bags, and stored in the dark at −80 °C until extraction. Preparation of Hydrophilic Extracts. The hydrophilic extract was obtained according to the method of Prior et al.26 with some modifications. The freeze-dried ground coffee (2.0 g) was weighed in a 50 mL Teflon tube, defatted three times with 20 mL of hexane, and centrifuged (Beckman Coulter Allegra 64R, Palo Alto, CA, USA) at 9000g for 15 min at 20 °C, and the supernatants were discarded. The solid residue was extracted three times with 20 mL of an acetone/ water/acetic acid solution (70:29.5:0.5, v/v/v). The extraction consisted of vortexing for 30 s followed by sonication for 4 min and finally centrifugation at 9000g for 15 min at 20 °C, collecting the supernatant. The three supernatants were combined in a roundbottom flask, and the organic solvent was removed in a rotary evaporator (T < 30 °C). The concentrated extract (aqueous phase) was frozen at −80 °C and freeze-dried for 120 h at −92 °C below 60 μmHg. After freeze-drying, the hydrophilic extract was transferred to an amber flask and stored at −80 °C. The yield of the hydrophilic extract was calculated according to eq 1:

excretion of caffeine and its derived methylxanthines and methyluric acids have been observed after consumption of a green/roasted coffee product.24 It is extremely important to take into account that the plasma antioxidant activity measured after the intake of coffee could be related to both the parent compounds and their metabolites.23 The bioactive compound profile of green coffee beans is mainly influenced by genetic aspects, such as species and variety, and by physiological aspects, such as maturity degree.25 For example, green coffee beans from C. canephora, C. liberica, C. congensis, C. eugenioides, C. stenophylla, C. racemosa, and C. kapakata present considerable variation in their chlorogenic acid (3.29−6.22 g/100 dry weight) and caffeine (0.96−2.12 g/100g dry weight) contents.3 Considering the importance of the genotype on the bioactive compound contents of coffee endosperm and consequently on the antioxidant capacity of the coffee extract, in this work we first evaluated the influence of coffee genotype on the bioactive compound qualitative and quantitative profiles. Second, we evaluated the in vitro scavenging capacity of coffee genotypes against the main biological relevant ROS and RNS, namely, peroxyl radical (ROO•), hydrogen peroxide (H2O2), hydroxyl radical (HO•), hypochlorous acid (HOCl), nitric oxide radical (NO•), and peroxynitrite anion (ONOO−). This is the first time that the capacity of green coffee to scavenge H2O2, HOCl, and NO • is reported. In addition, we estimated the contribution of each bioactive compound (5-caffeoylquinic acid, caffeic acid, trigonelline, caffeine, and theophylline) to the antioxidant capacity of the hydrophilic extracts from the 12 evaluated coffee genotypes.

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⎛ mass of hydrophilic extract (g) ⎞ yield (%, w/w) = ⎜ ⎟ × 100 ⎝ mass of freeze‐dried coffee (g) ⎠

MATERIALS AND METHODS

Reagents. Caffeine, 5-caffeoylquinic acid (5-CQA), caffeic acid, p-coumaric acid, ferulic acid, trigonelline, theobromine, theophylline, trolox, ascorbic acid, quercetin, cysteine, and rutin standards were acquired from Sigma-Aldrich (St. Louis, MO, USA), presenting a minimum of 95% of purity as determined by HPLC-DAD. HPLC grade methanol, glacial acetic acid, and formic acid were acquired from J. T. Baker (Phillipsburg, NJ, USA), and analytical grade formic acid was from Merck (Darmstadt, Germany). Folin−Ciocalteu reagent, fluorescein, α,α′-azodiisobutyramidine dihydrochloride (AAPH), lucigenin, 30% hydrogen peroxide solution (w/v), luminol, dihydrorhodamine 123 (DHR), N,N-dimethylformamide (DMF), sodium hypochlorite solution with 10−15% available chlorine, 4,5-diaminofluorescein (DAF-2), and 3-(aminopropyl)-1-hydroxy-3-isopropyl-2oxo-1-triazene (NOC-5) were acquired from Sigma-Aldrich. Sodium carbonate, sodium phosphate monobasic monohydrate, sodium phosphate dibasic, sodium hydroxide, sodium nitrite, sodium chloride, potassium chloride, and sodium carbonate were acquired from Synth (São Paulo, Brazil). Sodium phosphate tribasic dodecahydrate and 2-amino-2-(hydroxymethyl)-1,3-propanodiol (TRIS) were acquired from Sigma-Aldrich, ferrous chloride tetrahydrate was from J. T. Baker, and citric acid and ethylenediaminetetraacetic acid (EDTA) were from Quemis (Joinville, Brazil). Ultrapure water was obtained from a Millipore purification and filtration system (Billerica, MA, USA). Coffee Samples. Twelve green coffee samples belonging to four different species were evaluated: one C. kapakata; one C. racemosa; three cultivars of C. arabica, Catuaı ́ Vermelho IAC 144 (Caturra Amarelo IAC 476-11 × Mundo Novo IAC 374-19), IAC Ouro Verde (Catuaı ́ Amarelo IAC H2077-2-12-70 × Mundo Novo IAC 515-20), and Tupi IAC 1669-33 (Vila Sarchi × Hı ́brido de Timor CIFC 832/2); and seven varieties of C. canephora (Robusta IAC 3597, Robusta IAC 1655, Robusta IAC 1650, Kouilou IAC 70-14A, Kouilou IAC 70-14B, Laurentii, and Bukobensis). The term “genotype” will be used to denominate the different species, cultivars, and varieties of coffee throughout this paper. Eight of the 12 evaluated coffee

(1)

Determination of the Hydrophilic Extract Composition by HPLC-DAD and HPLC-DAD-MSn. The hydrophilic extract was suspended in a water/methanol (80:20, v/v) solution in the proportion of 5 mg of hydrophilic extract/1250 μL of solution, vortexed for 30 s, and centrifuged at 25000g for 10 min at 10 °C. The supernatant was filtered through a 0.45 μm membrane (Millipore, São Paulo, Brazil) and injected into a HPLC-DAD or HPLC-DAD-MSn. The quantitative analyses of the hydrophilic extract composition were carried out in a high-performance liquid chromatograph (HPLC, Shimadzu HPLC, Kyoto, Japan) equipped with a binary pump system (LC-10AD), helium gas degasser (DGU-2A), automatic injection system (SIL-10A), and diode array detector (DAD) (SPD-M10A). Trigonelline, theobromine, theophylline, caffeine, and the chlorogenic acids were separated on an ODS-C18 Shim-pack column (5 μm, 250 × 4.6 mm, Shimadzu) coupled to an ODS-C18 precolumn (5 μm, 4 × 3 mm, Phenomenex, Torrance, CA, USA), using a mixture of 80% (v/v) 10 mM citric acid (pH 2.5) and 20% (v/v) methanol (solvent A) and methanol (solvent B).27 The gradient was programmed as follows: 100:0 (solvent A/solvent B) for 19 min, from 100:0 to 80:20 in 10 min, then to 60:40 in 15 min, maintaining this condition for 10 min, changing to 100:0 in 1 min, and finaly maintaining this proportion for 9 min. The flow rate was 1.0 mL/min and the column temperature 30 °C. Trigonelline was quantified at 262 nm; theobromine, theophylline, and caffeine were quantified at 272 nm and the chlorogenic acids and caffeic acid at 325 nm. Quantitation was performed using analytical curves with six concentrations prepared in triplicate, and all of the analytical curves were linear (R2 = 0.99, p < 0.05). The limits of detection (LOD) and quantification (LOQ) were calculated on the basis of the parameters of the analytical curves (Table S1 of the Supporting Information). The chlorogenic acids and their derivatives were quantified using the area of the 5-CQA standard combined with B


Article


Figure 1. Chromatograms, obtained by HPLC-DAD, of the hydrophilic extract of C. canephora var. Kouilou IAC 70-14B recorded at 325 and 272 nm. Compound identification is shown in Table 1. the molar extinction coefficients as described by Farah et al.27 Due to the lack of a molar extinction coefficient for p-coumaroylquinic acid, this compound was quantified using the area of the 5-CQA standard. The cinnamoyl−amino acid conjugates were quantified using the molar extinction coefficient for 5-CQA as correction factor,28 and caffeoylferuloylquinic, p-coumaroylcaffeoylquinic, dimethoxycaffeoylquinic, and dimethoxyferuloylquinic acids were quantified using the mean of the molar extinction coefficient of the three dicaffeoylquinic acid isomers.29 The compounds present in the hydrophilic extract were identified using an HPLC (Shimadzu) equipped with a quaternary pump (LC-20AD), an online degasser (DGU-20A5), a Rheodyne valve with a 20 μL sample loop, a DAD detector (Shimadzu, SPD-M20A) connected to a mass spectrometer (MS) with an electrospray ionization source (ESI), and an ion trap analyzer (Bruker Daltonics, model Esquire 4000, Bremen, Germany). The chromatographic conditions were the same as described for quantification analyses, except that citric acid in solvent A was substituted by 0.3% formic acid (pH 2.5) because citric acid generates fragments at m/z 191, which interfere in compound identification. The mass spectra were acquired in the range from m/z 70 to 600, and the parameters used were as follows: ESI in the negative mode, capillary voltage of 2500 V, drying gas (N2) temperature of 310 °C and flow of 8 L/min, nebulizer = 30 psi, and the fragmentation energy of the MS2 and MS3 = 1.6 V. Trigonelline, theobromine, theophylline, caffeine, 5-CQA, and caffeic acid were identified on the basis of the combined results of the following parameters: elution order on reversed phase column, cochromatography with standards and characteristics of UV−visible and mass spectra as compared with those obtained with the standards analyzed under the same conditions. The other chlorogenic acids and their derivatives, for which no commercial standards were available, were tentatively identified on the basis of their elution order on the reversed phase column and characteristics of UV−visible and mass spectra (m/z of the deprotonated molecule, fragmentation pattern of the MS2 and MS3 spectra) as compared to those found in the literature.28,30−32 Assays to Determine the Reducing Capacity and ROS/RNS Scavenging Capacity. The hydrophilic extract was suspended in the specific buffer solution for each method in the proportion of 5 mg of hydrophilic extract/1250 μL of buffer, vortexed for 30 s, and centrifuged at 25000g for 10 min at 10 °C, and the supernatant was used in the reducing capacity and ROS/RNS scavenging capacity analyses. The assays were carried out in a microplate reader (Synergy Mx, Bio-Tek, Winooski, VT, USA) equipped with a thermostat and two

reagent dispensers. The following control assays were carried out for all microplates: (i) without the addition of the radical generator or reactive species, such that no interaction between the probe and the extract was observed, and (ii) inclusion of an analytical quality control (positive control), that is, a compound with antioxidant capacity known to scavenge the specific reactive species. Each assay corresponded to three experiments carried out in triplicate. The variation in the responses of the positive controls was below 15% during the assays. With the exception of the reducing capacity and the ROO• scavenging assay, the results were expressed in IC50 values, calculated by nonlinear regression analysis using the software GraphPad Prism 5.03. Reducing Capacity. The reducing capacity of the hydrophilic extract was determined using the Folin−Ciocalteu colorimetric method,33 which was adapted for analysis using a microplate reader.32 The reaction mixture contained the following reagents (final volume = 300 μL): hydrophilic extract dissolved in ultrapure water (33 and 73 μg/mL), Folin−Ciocalteu reagent (8.3%, v/v), and sodium carbonate solution (2.3%, w/v). The absorbance was monitored at 765 nm for 120 min at 25 °C, and quantification was done by way of an analytical curve for gallic acid in the range between 2 and 12.5 μg/mL, with the results expressed in milligrams of gallic acid equivalents per gram of extract (mg GAE/g). Peroxyl Radical Scavenging Assay. The ROO• was generated by the thermal decomposition of AAPH at 37 °C. The ROO• scavenging capacity was measured by monitoring the effect of the hydrophilic extract on the fluorescence decay due to fluorescein oxidation induced by ROO•.34,35 The reaction mixture contained fluorescein (61 nM), hydrophilic extract (five concentrations, 0.5− 5.2 μg/mL), and AAPH (19 mM) dissolved in 75 mM phosphate buffer (pH 7.4). The mixture was pre-incubated in the microplate reader during 10 min before AAPH addition. The fluorescence signal (excitation at 485 ± 20 nm and emission at 528 ± 20 nm) was monitored every minute until it reached 0.5% of the initial fluorescence signal. Quantification was carried out using an analytical curve for trolox in the range from 1 to 12 μmol, and the results were expressed in micromoles of trolox equivalents per gram of extract (μmol TE/g). Hydrogen Peroxide Scavenging Assay. The H2O2 scavenging capacity was measured by monitoring the effect of the hydrophilic extract on reducing the luminescence resulting from lucigenin oxidation induced by H2O2.34 The reaction mixture contained lucigenin (0.8 mM) and hydrophilic extract (five concentrations, 350−2400 μg/mL) dissolved in 50 mM Tris-HCl buffer (pH 7.4) and C


Article

Journal of Agricultural and Food Chemistry Table 1. Chromatographic and Mass Spectroscopy Characteristics of Chlorogenic Acids and Derivatives Identified in Hydrophilic Extracts from Coffee Beans, Obtained by HPLC-DAD-MSn peaka

a

tR (min)b

1

7.7

2

11.9

3

14.4

4

15.1

5

17.7

6 7

20.6 27.8

8

28.5

9

30.0

10

30.6

11

35.4

12

40.0

13

41.4

14

46.8

15

47.7

16

48.4

17

49.1

18

52.4

19

53.1

20

53.8

21

56.4

22

57.7

23

64.0

compound (abbrev)

[M − H]− (m/z)

3-caffeoylquinic acid (3-CQA) 3-p-coumaroylquinic acid (3-pCoQA) 3-feruloylquinic acid (3-FQA) 5-caffeoylquinic acid (5-CQA) 4-caffeoylquinic acid (4-CQA) caffeic acid (CA) 5-p-coumaroylquinic acid (5-pCoQA) 4-p-coumaroylquinic acid (4-pCoQA) 5-feruloylquinic acid (5-FQA) 4-feruloylquinic acid (4-FQA) caffeoyltyrosine (CTyr) 3,4-dicaffeoylquinic acid (3,4-diCQA) 3,5-dicaffeoylquinic acid (3,5-diCQA) 3-p-coumaroyl-4-caffeoylquinic acid (3pCo,4CQA) 3-caffeoyl-4-feruloylquinic acid (3C,4FQA) 4,5-dicaffeoylquinic acid (4,5-diCQA) 3-caffeoyl-5-feruloylquinic acid (3C,5FQA) caffeoyltryptophan (CTrp) 4-feruloyl-5-caffeoylquinic acid (4F,5CQA) 4-caffeoyl-5-feruloylquinic acid (4C,5FQA) p-coumaroyltryptophan (p-CoTrp) 4-dimethoxycinnamoyl-5-caffeoylquinic acid (4D,5CQA) 4-dimethoxycinnamoyl-5-feruloylquinic acid (4D,5FQA)

353 337 367 353 353 179 337 337 367 367 342 515 515 499 529 515 529 366 529 529 350 543 557

fragments (m/z)c 2

MS MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3 MS2 MS3

identity confirmation

[353]: 191, 179, 135, 173 [353→191]: 127, 173, 85, 93 [337]: 163 [337→163]: 119 [367]: 193, 133, 173 [367→193]: 133, 149 [353]: 191, 179 [353→191]: 127, 173, 85, 111 [353]: 173, 179, 191, 135 [353→173]: 93, 111, 155, 71 [179]: 135 [337]: 191, 163 [337→191]: 127, 172, 85 [337]: 173, 191, 163 [337→173]: 93, 111, 155 [367]: 191, 173 [367→191]: 127, 173, 85, 93 [367]: 173, 191 [367→173]: 93, 111, 155 [342]: 207, 163, 135, 119 [342→207]: 163, 119 [515]: 353, 173, 179, 191, 335 [515→353]: 173, 179, 191, 135 [515]: 353, 191 [515→353]: 191, 179, 135, 173 [499]: 353, 335, 337, 173, 179 [499→353]: 173, 179, 135, 191 [529]: 367, 173, 335, 193 [529→367]: 173, 193 [515]: 353, 173, 203 [515→353]: 173, 179, 191, 135 [529]: 353, 367, 191 [529→353]: 191, 179, 135 [366]: 229, 135, 185 [366→229]: 185, 100, 142 [529]: 367, 173 [529→367]: 173, 193, 134 [529]: 353, 367, 173, 191 [529→353]: 173, 179, 191, 135 [350]: 229, 185 [350→229]: 185, 100, 142 [543]: 381, 367, 173, 207, 349, 335 [543→381]: 173, 207 [557]: 381, 349, 207, 173 [557→381]: 173, 207

30 30 30 analytical standard 30 standard 30 30 30 30 28 30 30 32 30 30 30 28 30 30 28 31 31

Numbered according to the chromatogram shown in Figure 1. bRetention time on C18 column. cThe base peaks are shown in bold. extract on reducing the fluorescence resulting from the oxidation of DHR to rhodamine 123 induced by HOCl.34 The HOCl was prepared by adjusting the pH of a 1% NaOCl (w/v) solution to 6.2 with 10% H2SO4 (v/v). The HOCl concentration was determined spectrophotometrically at 235 nm using the molar absorption coefficient of 100 M−1 cm−1, and the other dilutions were made in 100 mM phosphate buffer (pH 7.4). The reaction mixture contained hydrophilic extract (five concentrations, 0.7−71 μg/mL), DHR (5 μM), and HOCl (5 μM) dissolved in 100 mM phosphate buffer (pH 7.4). The fluorescence signal (excitation at 485 ± 20 nm and emission at 528 ± 20 nm) was measured immediately after the addition of HOCl at 37 °C. Nitric Oxide Scavenging Assay. NO• was generated by thermal decomposition of NOC-5. The NO• scavenging capacity was

1% H2O2 (v/v). The chemiluminescence signal was measured after 5 min of incubation at 37 °C. Hydroxyl Radical Scavenging Assay. The HO• was generated by the Fenton reaction (FeCl2−EDTA−H2O2). The HO• scavenging capacity was measured by monitoring the effect of the hydrophilic extract on reducing the luminescence resulting from luminol oxidation induced by HO•.34 The reaction mixture contained hydrophilic extract (five concentrations, 0.4−8.0 μg/mL) dissolved in ultrapure water, luminol (20 μM) in 0.5 M carbonate buffer (pH 10), FeCl2−EDTA (25 μM, 100 μM) solution in ultrapure water, and H2O2 (3.5 mM). The chemiluminescence signal was measured after 5 min of incubation at 37 °C. Hypochlorous Acid Scavenging Assay. The HOCl scavenging capacity was measured by monitoring the effect of the hydrophilic D


Article

The results correspond to the average of triplicate ± standard deviation, expressed as mg/100 g of hydrophilic extract. Means with different letters in the same column are significantly different (p < 0.05). CQA total, caffeoylquinic acid (sum of 3-CQA, 4-CQA, and 5-CQA). cFQA total, feruloylquinic acid (sum of 3-FQA, 4-FQA, and 5-FQA). dp-CoQA total, p-coumaroylquinic acid (sum of 3-pCoQA, 4pCoQA, and 5-pCoQA). edi-CQA total, dicaffeoylquinic acid (sum of 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA). fCFQA total, caffeoylferuloylquinic acid (sum of 3C,4FQA, 3C,5FQA, 4F,5CQA, and 4C,5FQA). gCGA, total chlorogenic acid (sum of CQA, FQA, p-CoQA, di-CQA, CFQA, p-coumaroylcaffeoylquinic acid, and dimethoxycinnamoylquinic acid). hTCTrp, cinnamoyl−amino acid conjugates (sum of caffeoyltyrosine, caffeoyltryptophan, and p-coumaroyltryptophan). ind, not detected (limit of detection, caffeic acid = 0.09 μg/mL).

measured by monitoring the effect of the hydrophilic extract in the reduction of the fluorescence resulting from the oxidation of 4,5diaminofluoresceine (DAF-2) to triazolofluoresceine induced by NO•.34 The reaction mixture contained DAF-2 (5 μM), hydrophilic extract (five concentrations, 0.7−85 μg/mL), and NOC-5 (60 μM) dissolved in 50 mM phosphate buffer (pH 7.4). The fluorescence signal (excitation at 485 ± 20 nm and emission at 528 ± 20 nm) was measured after 30 min of incubation at 37 °C. Peroxynitrite Anion Scavenging Assay. The ONOO− scavenging capacity was measured by monitoring the effect of the hydrophilic extract on the reduction of the fluorescence resulting from the oxidation of DHR to rodamine 123 induced by ONOO−.34 The ONOO− was synthesized as previously described by Gomes et al.36 The reaction mixture contained DHR (5 μM) in phosphate buffer (90 mM NaCl, 50 mM Na3PO4, 5 mM KCl, pH 7.4), the hydrophilic extract (five concentrations, 0.3−35 μg/mL) dissolved in phosphate buffer, and ONOO− (600 nM). The fluorescence signal (excitation at 485 ± 20 nm and emission at 528 ± 20 nm) was measured after 5 min of incubation at 37 °C. To simulate the physiological CO 2 concentration, an assay was carried out in parallel in the presence of 25 mM NaHCO3. Contribution of Each Bioactive Compound to the Antioxidant Capacity. The contribution of each bioactive compound (5-CQA, caffeic acid, trigonelline, caffeine, and theophylline) to the antioxidant capacity of the hydrophilic extract was calculated considering the antioxidant capacity of the extract, the antioxidant capacity of the analytical standard of each bioactive compound previously evaluated by Rodrigues et al.,34 and the concentration of each bioactive compound present in the extract. In the assays of reducing capacity and ROO• scavenging capacity, the percent contribution (a) was calculated using eq 2. In the assays of H2O2, HO•, HOCl, NO•, and ONOO− scavenging capacity, for which the antioxidant capacity was expressed as the IC50, the following steps were used to calculate the percent contribution (b): (1) calculation of the concentration of the bioactive compound in the fraction of the hydrophilic extract responsible for inhibiting 50% of the probe oxidation (eq 3) and calculation of the decimal logarithm of this concentration; (2) calculation of the percent inhibition of the bioactive compound present in the extract using the equation of the dose− response curve obtained for each analytical standard (eq 4). % contribution (a) [concentration] × reducing capacity of the standard = reducing capacity of the extract concentration (μg/mL) =

[concentration] × IC50 of extract 100

% contribution (b) = 100/1 + 10(logIC50 − X ) × Hill slope

(2) (3) (4)

where [concentration] = concentration of the bioactive compound in the extract (g/100 g), log IC50= logarithm of the IC50 of the analytical standard, and X = logarithm of the concentration of the bioactive compound present in the extract. Statistical Analysis. The results obtained for the contents of bioactive compounds, reducing capacity, and ROS/RNS scavenging capacities of the hydrophilic extracts were submitted to an analysis of variance (ANOVA), and the means were compared by Tukey’s test using Origin 8 software. The similarity of the hydrophilic extracts with respect to reducing capacity and scavenging capacity against ROS/ RNS was determined using the method of hierarchical clustering by nonpondered average and the Euclidean distance as similarity index. The principal component analysis was applied to characterize the hydrophilic extracts according to their reducing capacity, scavenging capacity against ROS/RNS, and the content of bioactive compounds using Statistica 6.0 software. The reducing capacity and ROS/RNS scavenging capacity were used as the active variables. The chlorogenic acid (sum of caffeoylquinic, feruloylquinic, p-coumaroylquinic, dicaffeoylquinic, caffeoylferuloylquinic, p-coumaroylcaffeoylquinic, dimethoxycaffeoylquinic, and dimethoxyferuloylquinic acids), cinnamoyl−amino

b

a

0.2a 0.3c 0.2cd 0.1cd 0.2ef 0.3f 0.4b 0.2ef 0.2de 0.5c 0.3f ± ± ± ± ± ± ± ± ± ± ± 0.8f 1.0f 5e 28d 20cd 16c 28c 33b 26a 17c ± ± ± ± ± ± ± ± ± ±

caffeic acid

nd 9.9 6.3 5.7 5.9 4.3 4.2 7.5 4.5 5.1 6.2 4.2 ndi nd 26.5 27.0 101 672 741 758 745 1026 1116 768 225de 595g 536g 463g 194g 1111f 979cde 324bcd 1175ef 799a 523bc 393ab ± ± ± ± ± ± ± ± ± ± ± ± 33391 24693 22869 23854 25183 30397 33276 35172 32421 37965 35593 36824 3e 4f 3f 2.4f 4f 59a 41b 6bc 35d 39a 31bc 49ab ± ± ± ± ± ± ± ± ± ± ± ± 647 126 117 94.5 109 1441 1273 1218 951 1449 1150 1162 21c 112e 78f 58f 107f 177c 159d 79b 163e 104d 137a 81d ± ± ± ± ± ± ± ± ± ± ± ± C. C. C. C. C. C. C. C. C. C. C. C.

kapakata racemosa arabica cv. Catuaı ́ Vermelho IAC 144 arabica cv. IAC Ouro Verde arabica cv. Tupi IAC 1669-33 canephora var. Robusta IAC 3597 canephora var. Robusta IAC 1655 canephora var. Robusta IAC 1650 canephora var. Kouilou IAC 70-14A canephora var. Kouilou IAC 70-14B canephora var. Laurentii canephora var. Bukobensis

24.1 23.9 22.4 22.6 25.1 26.3 23.6 24.1 28.4 28.9 25.1 29.0

25709 19863 17929 19066 20030 18866 19054 22117 21663 24844 23124 25413

± ± ± ± ± ± ± ± ± ± ± ±

194a 457d 409e 369de 54d 687de 544de 196bc 766c 483a 290b 288a

1829 803 1696 1382 1748 4985 8189 5859 5686 6744 4698 5230

12fg 14i 39gh 26h 16gh 170de 217a 50c 204c 159b 61e 30d

231 185 284 273 366 113 120 70.6 185 170 234 183

3c 9d 6b 6b 11a 4e 3e 0.6f 6d 4d 4c 5d ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4951 3708 2792 2994 2869 4929 4574 5850 3859 4245 6326 4565

TCTrp totalh CGAg CFQA totalf di-CQA totale p-CoQA totald FQA totalc CQA totalb yield (%, w/w) hydrophilic extract origina

Table 2. Yield and Contents of the Main Chlorogenic Acid Subgroups (CQA, FQA, p-CoQA, di-CQA, and CFQA), Total Chlorogenic Acids (CGA), Cinnamoyl−Amino Acid Conjugates (TCTrp), and Caffeic Acid in the Hydrophilic Extracts of the Different Coffee Genotypes


E


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Table 3. Trigonelline, Theobromine, Theophylline, and Caffeine Contents in the Hydrophilic Extracts of the Different Coffee Genotypes hydrophilic extract origina C. C. C. C. C. C. C. C. C. C. C. C.

kapakata racemosa arabica cv. Catuaı ́ Vermelho IAC 144 arabica cv. IAC Ouro Verde arabica cv. Tupi IAC 1669-33 canephora var. Robusta IAC 3597 canephora var. Robusta IAC 1655 canephora var. Robusta IAC 1650 canephora var. Kouilou IAC 70-14A canephora var. Kouilou IAC 70-14B canephora var. Laurentii canephora var. Bukobensis

trigonelline 6712 5438 4719 4578 4436 3838 4934 4686 4019 3952 3922 3091

± ± ± ± ± ± ± ± ± ± ± ±

theobromine

theophylline

ndb 43.2 nd nd 3.9 nd nd nd 4.6 4.9 nd nd

7.8 3.3 nd nd nd nd 11.5 9.3 nd nd 12.2 nd

70a 105b 101cd 69de 62e 151f 120c 28cde 133f 67f 54f 25g

± 1.3a

± 0.1b

± 0.4b ± 0.2b

caffeine

± 0.3c ± 0.03d

3923 4780 4616 4163 4482 6600 8937 9321 9963 11776 9467 9210

± 0.5a ± 0.2b

± 0.5a

± ± ± ± ± ± ± ± ± ± ± ±

46g 54e 112ef 84fg 24efg 265d 207c 71c 299b 402a 125bc 153c

The results correspond to the average of triplicate ± standard deviation, expressed as mg/100 g of hydrophilic extract. Means with different letters in the same column are significantly different (p < 0.05). bnd, not detected (limit of detection, theobromine = 0.07 μg/mL; theophylline = 0.04 μg/mL). a

Table 4. Peroxyl Radical (ROO•), Hydrogen Peroxide (H2O2), Hydroxyl Radical (HO•), and Hypochlorous Acid (HOCl) Scavenging Capacities and Reducing Capacity of the Hydrophilic Extracts from Different Coffee Genotypes and Positive Controls IC50a (μg/mL) hydrophilic extracts origin C. kapakata C. racemosa C. arabica cv. Catuaı ́ Vermelho IAC 144 C. arabica cv. IAC Ouro Verde C. arabica cv. Tupi IAC 1669-33 C. canephora var. Robusta IAC 3597 C. canephora var. Robusta IAC 1655 C. canephora var. Robusta IAC 1650 C. canephora var. Kouilou IAC 70-14a C. canephora var. Kouilou IAC 70-14B C. canephora var. Laurentii C. canephora var. Bukobensis positive controls ascorbic acid quercetin gallic acid cysteine

reducing capacityb

ROO•c

H2O2

HO•

HOCl

205 ± 14bcd 152 ± 10e 145 ± 11e 149 ± 8e 147 ± 9e 192 ± 10d 202 ± 11cd 215 ± 10abc 206 ± 14bcd 225 ± 12a 219 ± 12ab 226 ± 14a

3738 ± 246cd 2643 ± 190g 2453 ± 161g 3008 ± 261ef 2700 ± 231fg 3204 ± 220e 4111 ± 404bc 3689 ± 276d 4230 ± 350b 4735 ± 370a 4020 ± 322bcd 3681 ± 246d

877 ± 31b 1083 ± 27ef 1526 ± 19g 1133 ± 20f 863 ± 33b 941 ± 4c 1022 ± 24de 984 ± 8cd 831 ± 17ab 779 ± 25a 868 ± 16b 824 ± 9ab

1.49 ± 0.04cd 2.15 ± 0.10f 2.96 ± 0.07h 2.76 ± 0.06g 2.82 ± 0.07gh 1.77 ± 0.06e 1.61 ± 0.01d 1.56 ± 0.03d 1.39 ± 0.03bc 1.33 ± 0.05ab 1.33 ± 0.01ab 1.25 ± 0.01a

4.26 ± 0.28a 5.08 ± 0.33a 16.07 ± 0.19e 11.96 ± 0.51d 13.22 ± 0.77d 7.47 ± 0.35b 10.60 ± 0.40c 9.72 ± 0.56c 9.88 ± 0.34c 15.26 ± 0.87e 9.57 ± 0.40c 14.62 ± 0.26e

721 ± 45

155 ± 18 28032 ± 2025 0.16 ± 0.01 66.46 ± 6.37

a

IC50 = in vitro inhibitory concentration necessary to decrease by 50% the oxidative effect of the reactive species in the tested media (mean of triplicate ± standard deviation). Means with the different letters in the same column are significantly different (p < 0.05). bMilligrams of gallic acid equivalents per gram of hydrophilic extract or gram of standard compound (mean of triplicate ± standard deviation). cMicromoles of trolox equivalents per gram of hydrophilic extract or gram of standard compound (mean of triplicate ± standard deviation).

derivative by HPLC-DAD-MSn in green coffee beans was previously presented in the literature, and the references for these papers can be found in Table 1. The UV−visible spectra of the compounds identified in hydrophilic coffee extracts are presented in Figure S1 of the Supporting Information. The nomenclature of all chlorogenic acid isomers cited in the present work was based on the recommended IUPAC numbering system.37 In general, the qualitative profiles of the bioactive compounds were similar in all of the coffee genotypes (Tables 2 and 3), and the differences occurred in relation to the concentrations of these compounds in the extracts. On average, the hydrophilic extracts of C. canephora (34.5 g/100 g) and C. kapakata (33.4 g/100 g) showed 30% more chlorogenic acids

acid conjugates (sum of caffeoyltyrosine, caffeoyltryptophan, and p-coumaroyltryptophan), caffeic acid, trigonelline, theobromine, theophylline, and caffeine contents were used as the supplementary variables in the derivation of the principal components, being projected in the same space.

■

RESULTS Bioactive Compounds in the Hydrophilic Extracts. Figure 1 shows a typical chromatogram of the separation of trigonelline, theobromine, caffeine, and chlorogenic acids and derivatives in the hydrophilic coffee extract. A total of 23 hydroxycinnamic derivatives were identified and quantified in the hydrophilic extracts (Table 1), 19 being chlorogenic acids, 3 cinnamoyl−amino acid conjugates, and 1 free cinnamic acid. A detailed description of the identification of each hydroxycinnamic F


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Table 5. Nitric Oxide (NO•) and Peroxynitrite Anion (ONOO−) Scavenging Capacities of the Hydrophilic Extracts from Different Coffee Genotypes and Positive Controls

than those of C. arabica (24 g/100 g) and C. racemosa (24.7 g/ 100 g) (Table 2). In addition to the differences in total chlorogenic acid contents, differences were found in the contributions of the subgroups of the compounds making up the chlorogenic acids. For example, the subgroups CQA, FQA, p-CoQA, di-CQA, and CFQA represented, on average, 79, 6.7, 1.3, 12, and 0.4% of the total chlorogenic acid contents of the C. arabica extracts, whereas they represented, on average, 64, 17, 0.4, 14, and 3.6% of the C. canephora extracts. Among the chlorogenic acid subgroups, the elevated contents of FQA and CFQA in the C. canephora extracts stood out, being, on average, 4 and 12 times higher than the respective values found in the C. arabica extracts. On the other hand, the C. arabica extracts contained, on average, twice the amount of p-CoQA found in the C. canephora extracts. In general, the hydrophilic extracts presented 5-CQA, 5-FQA, 5-pCoQA, 3,5-diCQA, and 3C,5FQA as the majority isomers of each subgroup (Tables S2 and S3 of the Supporting Information). The cinnamoyl−amino acid conjugates were not found in the hydrophilic extracts of C. kapakata or C. racemosa, and the contents found in C. canephora (832 mg/100 g) were, on average, 16 times higher than those found in C. arabica (51 mg/100 g) (Table 2). Caffeine was the major alkaloid found in the hydrophilic extracts of all the coffee genotypes studied (Table 3). C. racemosa beans presented a distinct behavior, because in addition to caffeine, which was the major alkaloid, the theobromine contents were, on average, 10 times higher than those found in C. arabica (Tupi cultivar) and C. canephora (Kouilou varieties). The hydrophilic extracts of C. canephora presented twice the amounts of caffeine found in the other coffee species. On the other hand, C. kapakata and C. racemosa stood out for their elevated contents of trigonelline. Reducing Capacity and ROS/RNS Scavenging Capacity of the Hydrophilic Extracts. The 12 hydrophilic coffee extracts presented reducing capacity and were able to scavenge all of the evaluated ROS (ROO•, H2O2, HO•, and HOCl) (Table 4) and RNS (NO• and ONOO−) (Table 5), always in a dose-dependent manner and with IC50 values to the order of micrograms per milliliter. On average, the reducing capacity and ROO•, H2O2, HO•, NO•, and ONOO− scavenging capacities of the hydrophilic extracts were, respectively, 3, 8, 6, 12, 17, and 8 times lower than that of the positive control used in each assay. On the other hand, the capacity of the hydrophilic extracts to scavenge HOCl was, on average, 6 times higher than that of the cysteine standard used as positive control. The capacity of the hydrophilic extracts to scavenge ONOO− was also evaluated in the presence of NaHCO3 with the objective of simulating the physiological CO2 concentrations. Under this condition, the CO2 could modulate the reactivity of ONOO− due to the rapid reaction between these two compounds, generating nitrogen dioxide radical (NO2•) and the carbonate radical anion (CO3•−), which are the main radicals responsible for the oxidation and nitration reactions observed in vivo.38 In general, the hydrophilic extracts maintained their IC50 values in the assays carried out in the presence of NaHCO3 (Table 5), indicating that they are also effective in scavenging NO2• and CO3•−. The antioxidant capacity of the hydrophilic coffee extracts was influenced both by the coffee genotype and by the evaluated reactive species. When the cluster analysis was applied using the reducing capacity and ROS/RNS scavenging capacity

IC50A (μg/mL) NO•

hydrophilic extracts C. kapakata C. racemosa C. arabica cv. Catuaı ́ Vermelho IAC 144 C. arabica cv. IAC Ouro Verde C. arabica cv. Tupi IAC 1669-33 C. canephora var. Robusta IAC 3597 C. canephora var. Robusta IAC 1655 C. canephora var. Robusta IAC 1650 C. canephora var. Kouilou IAC 70-14A C. canephora var. Kouilou IAC 70-14B C. canephora var. Laurentii C. canephora var. Bukobensis positive controls rutin trolox

ONOO− absence of NaHCO3

presence of 25 mM NaHCO3

9.96 ± 0.81c 13.90 ± 0.94d 17.62 ± 0.51ef

1.20 ± 0.09ab 1.94 ± 0.08ef 2.36 ± 0.09g

1.43 ± 0.06a 2.13 ± 0.07b 3.06 ± 0.24c

15.57 ± 0.84de

1.86 ± 0.12ef

2.20 ± 0.14b

18.26 ± 1.25f

2.00 ± 0.16f

2.21 ± 0.14b

6.86 ± 0.55a

1.68 ± 0.04de

1.99 ± 0.10b

6.11 ± 0.38a

1.40 ± 0.10bc

1.39 ± 0.08a

7.35 ± 0.34ab

1.57 ± 0.05cd

2.09 ± 0.15b

9.41 ± 0.57bc

1.28 ± 0.06abc

1.52 ± 0.15a

9.18 ± 0.74bc

1.08 ± 0.05a

1.21 ± 0.08a

6.40 ± 0.56a

1.09 ± 0.03a

1.28 ± 0.08a

6.77 ± 0.60a

1.10 ± 0.03a

1.24 ± 0.07a

0.20 ± 0.005

0.20 ± 0.01

0.78 ± 0.05

A

IC50 = in vitro inhibitory concentration necessary to decrease by 50% the oxidative effect of the reactive species in the tested media (mean of triplicate ± standard deviation). Means with the different superscript letters at the same column are significantly different (p < 0.05).

of the hydrophilic extracts as the variables, two large groups were formed (Figure 2). The first group was formed by the three C. arabica extracts and the C. racemosa extract. The second group was formed by the seven C. canephora extracts and the C. kapakata extract. The same two groups formed in the cluster analysis were also formed in the principal components analysis. The first group, formed by the three C. arabica extracts and the C. racemosa extract, was located to the right of the first principal component (PC1), and the second, formed by the seven C. canephora extracts and the C. kapakata extract, was to the left of PC1 (Figure 3c). The hydrophilic extracts from the second group presented higher reducing capacity and higher scavenging capacity against ROO•, H2O2, HO•, NO•, and ONOO− than the hydrophilic extracts from the first group (Figure 3a,c). The higher capacity to scavenge these five reactive species was positively correlated with chlorogenic acid, cinnamoyl−amino acid conjugate, and caffeine contents (Figure 3b). Estimate of the Contribution of Each Bioactive Compound to the Antioxidant Capacity. 5-CQA was mainly responsible for the reducing capacity and ROS/RNS scavenging capacity of the hydrophilic extracts, with the exception of HOCl (Table 6), among the five analytical standards of bioactive compounds present in the evaluated G


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Figure 2. Dendrogram obtained by hierarchical cluster analysis of the reducing capacity and ROS/RNS scavenging capacity of hydrophilic extracts. Rac, C. racemosa; Tupi, C. arabica cv. Tupi IAC 1669-33; OV, C. arabica cv. IAC Ouro Verde; Cat, C. arabica cv. Catuaı ́ Vermelho IAC 144; Kap, C. kapakata; 14B, C. canephora var. Kouilou IAC 70-14B; 14A, C. canephora var. Kouilou IAC 70-14A; Lau, C. canephora var. Laurentii; Buk, C. canephora var. Bukobensis; 1655, C. canephora var. Robusta IAC 1655; 1650, C. canephora var. Robusta IAC 1650; 3597, C. canephora cv. Robusta IAC 3597.

recognized antioxidant capacity against ROS and RNS IC50 values cited by Rodrigues et al.34). The hydrophilic extracts from the seven C. canephora varieties and from C. kapakata stood out for their elevated reducing capacities and capacities to scavenge ROO•, H2O2, HO•, NO•, and ONOO− (Figure 3). These green coffee hydrophilic extracts presented higher capacities to scavenge ROO• (3.20−4.74 μmol TE/mg), HO• (IC50 = 1.25−1.77 μg/mL), NO• (IC50 = 6.11−9.96 μg/mL), and ONOO− (IC50 = 1.08− 1.68 μg/mL) than mana-cubiu pulp extracts (ROO•, 0.32 μmol TE/mg; HO•, 36 μg/mL; ONOO−, 20 μg/mL)41 and artichoke leaf extracts (NO•, 5.5−11.0 μg/mL; ONOO−, 3.4−5.1 μg/mL),42 5-CQA being the main phenolic compound identified in both of these extracts. The greater scavenging capacity of the green coffee hydrophilic extracts was probably related to their higher 5-CQA concentration (15−23%, w/w) when compared to mana-cubiu (0.4%, w/w) and artichoke (4.0−6.4%, w/w) extracts. In addition, the study of the estimated contribution of each bioactive compound to the antioxidant capacity showed that 5-CQA was the main component responsible for the scavenging capacity of the hydrophilic extracts against ROS and RNS (Table 6). In fact, 5-CQA was the major phenolic compound found in coffee, corresponding respectively to 52 and 68% of the total chlorogenic acids present in the hydrophilic extracts of C. canephora and C. kapakata (Table S2 of the Supporting Information). This group of results indicates 5-CQA as the main contributor to the reducing capacity and ROO•, HO•, NO•, and ONOO− scavenging capacities of coffee hydrophilic extracts. Some studies have reported the mechanism by which phenolic compounds such as 5-CQA scavenge some of these reactive species. Phenolic compounds can scavenge ROO• and HO• by transfer of hydrogen atoms or electrons and by addition to double bonds.43,44 The three mechanisms can occur, but the ROO• scavenging by phenolic compounds occurs mainly due to the transference of a hydrogen atom.35

hydrophilic extracts. Caffeic acid, trigonelline, caffeine, and theophylline did not contribute to the ROS/RNS scavenging capacity of the extracts, with the exception of the contribution of trigonelline to scavenge H2O2 (2.4−14.6%) and of caffeic acid to scavenge HOCl (1.5−2.0%).

■

DISCUSSION Studies carried out with green coffee beans of the two main species cultivated in the world, C. arabica and C. canephora, showed that the C. canephora beans contained higher amounts of chlorogenic acids, cinnamoyl−amino acid conjugates, and caffeine.29,39,40 The results of the present study confirmed this information and also showed that the quantitative profile of each chlorogenic acid subgroup was also an important factor in discriminating between C. arabica, C. canephora, C. kapakata, and C. racemosa beans. The chlorogenic acid subgroups that aided in the discrimination between the coffee species evaluated in this study were caffeoylferuloylquinic and feruloylquinic acids (both with higher contents in C. canephora beans) and p-coumaroylquinic acid (with a higher content in C. arabica beans). In addition, cinnamoyl−amino acid conjugates and caffeine (both with higher contents in C. canephora beans) and trigonelline (with a higher content in C. kapakata and C. racemosa beans) also contributed to differentiating among the species (Tables 2 and 3). The quantitative profile of the bioactive compounds was also used to explain the high antioxidant capacity of the hydrophilic extracts from different coffee genotypes against ROO•, H2O2, HO•, HOCl, NO•, and ONOO− and possibly against NO2• and CO3•− (indirectly evaluated reactive species). The hydrophilic extracts of coffee could be considered potent antioxidants due to (i) the low IC50 values required to scavenge the reactive species, to the order of micrograms per milliliter, and (ii) the similar or higher antioxidant capacity when compared to coffee brews and fruit and plant extracts (with H


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Figure 3. Principal component analysis of the reducing capacity and ROS/RNS scavenging capacity of hydrophilic extracts: (a) active variable projection; (b) supplementary variable projection; (c) scatter plot for the hydrophilic extracts. CR, reducing capacity; ROO•, peroxyl radical; H2O2, hydrogen peroxide; HO•, hydroxyl radical; HOCl, hypochlorous acid; NO•, nitric oxide; ONOO−, peroxynitrite anion; CGA, chlorogenic acids; TCTrp, cinnamoyl−amino acid conjugates (caffeoyltyrosine, caffeoyltryptophan, and p-coumaroyltryptophan); CA, caffeic acid; Trig, trigonelline; Caf, caffeine; Teobr, theobromine; Teof, theophylline; Rac, C. racemosa; Tupi, C. arabica cv. Tupi IAC 1669-33; OV, C. arabica cv. IAC Ouro Verde; Cat, C. arabica cv. Catuaı ́ Vermelho IAC 144; Kap, C. kapakata; 14B, C. canephora var. Kouilou IAC 70-14B; 14A, C. canephora var. Kouilou IAC 70-14A; Lau, C. canephora var. Laurentii; Buk, C. canephora var. Bukobensis; 1655, C. canephora var. Robusta IAC 1655; 1650, C. canephora var. Robusta IAC 1650; 3597, C. canephora cv. Robusta IAC 3597.

The literature has also reported the ONOO− scavenging capacity of phenolic compounds, which can occur by two mechanisms depending on the structural characteristcs of the phenolic compound. Monophenols, such as feruloylquinic and p-coumaroylquinic acids, act preferably by nitration, and the catechols, such as the caffeic, caffeoylquinic, and dicaffeoylquinic acids and the cinnamoyl−amino acid conjugates, act by electron donation.45 On the other hand, the elevated reducing capacity of the hydrophilic extracts is related to the electron transfer mechanism carried out by the phenolic compounds. The study of reducing capacity is important because some ROS and RNS can be scavenged after they receive one or more electrons. The compounds 5-CQA, 3-CQA, and 4-CQA are formed by the esterification of a caffeic acid molecule with quinic acid and

present a catechol in their structure. The catechol group of the phenolic compounds has an important role in scavenging ROS and RNS, because it can act in the transference of hydrogen atoms and electrons. Other compounds containing catechol groups in their structures are also present in the green coffee hydrophilic extracts, such as dicaffeoylquinic acids, caffeoylferuloylquinic acid, caffeoyltyrosine, and caffeoyltryptophan. Within this group, the dicaffeoylquinic acids stand out because they present two catechol groups in their structure, conferring a high antioxidant capacity with respect to the radical DPPH and the superoxide anion radical.46 This structural characteristic of the dicaffeoylquinic acids allied to an elevated concentration in the hydrophilic C. canephora and C. kapakata extracts (Table 2) probably contributed to the high reducing capacity and ROO•, I


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Table 6. Estimate (Percent) of the Contribution of the Standards of Each Bioactive Compound on the Reducing Capacity and ROS and RNS Scavenging Capacity of the Hydrophilic Coffee Extracts scavenging capacity assay

5-CQA

caffeic acid

trigonelline

caffeine

theophylline

reducing capacity ROO• H2O2 a HO• a HOCla NO• a ONOO−a a ONOO−p a

53−74 47−74 15−26 18−41 0.7−2.9 9−25 36−48 33−49

0.03−0.07 0.03−0.08 NC NC 1.5−2.0 0.08−0.39 0.06−0.22 0.004−0.02

NC NC 2.4−14.6 NC NC NC NC NC

0.1−0.4 0.01−0.02 NC NC NC NC NC NC

NC NC NC NC NC NC NC NC

a

The estimate is based on concentration necessary to decrease by 50% the oxidative effect of the reactive species in the tested media (IC50). NC, no contribution; ROO•, peroxyl radical; H2O2, hydrogen peroxide; HO•, hydroxyl radical; HOCl, hypochlorous acid; NO•, nitric oxide; ONOO−a, peroxynitrite anion in the absence of NaHCO3; ONOO−p, peroxynitrite anion in the presence of 25 mM NaHCO3; 5-CQA, 5-caffeoylquinic acid.

H2O2, HO•, NO•, and ONOO− scavenging capacities of these extracts. The hydrophilic extracts were potent HOCl scavengers, presenting a scavenging capacity on average 6 times higher than that of cysteine, the positive control used in the method. Despite contributing

Influence of Coffee Genotype on Bioactive Compounds and the in

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