Article pubs.acs.org/JAFC
Influence of the Stage of Ripeness on the Composition of Iridoids and Phenolic Compounds in Genipap (Genipa americana L.) Adria de Sousa Bentes and Adriana Zerlotti Mercadante* Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), 13083-862 Campinas, SP, Brazil ABSTRACT: Genipap fruits, native to the Amazon region, were classified in relation to their stage of ripeness according to firmness and peel color. The influence of the part of the genipap fruit and ripeness stage on the iridoid and phenolic compound profiles was evaluated by HPLC-DAD-MSn, and a total of 17 compounds were identified. Geniposide was the major compound in both parts of the unripe genipap fruits, representing >70% of the total iridoids, whereas 5-caffeoylquinic acid was the major phenolic compound. In ripe fruits, genipin gentiobioside was the major compound in the endocarp (38%) and no phenolic compounds were detected. During ripening, the total iridoid content decreased by >90%, which could explain the absence of blue pigment formation in the ripe fruits after their injury. This is the first time that the phenolic compound composition and iridoid contents of genipap fruits have been reported in the literature. KEYWORDS: secondary metabolites, Genipa americana L., ripening stages, tropical fruit, HPLC-DAD-MSn
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INTRODUCTION Iridoids are secondary plant metabolites of angiosperms and constitute the largest group of monoterpenoids with a cyclopentan-[C]-pyran skeleton.1 These compounds can be classified as (Figure 1) (A) simple or nonglycosylated; (B)
Genipin, 13, can be obtained from the hydrolysis of the geniposide, 9 (Figure 2), which is its glycosylated form, by the action of the enzyme β-glucosidase,11 Geniposide, 9, has already been found in genipap fruits (Genipa americana L.) and in cape jasmine (Gardenia jasminoides), both plants from the Rubiaceae family.14−16 Genipap is a fruit native to the Amazon region, which can also be found in other tropical and subtropical regions in Latin America.17 Genipap fruits in the unripe stage are widely used by various ethnic groups of Brazilian Indians to obtain the blue pigment by exposing the inside of the fruits to air. The Indians use the blue pigment to paint their bodies and ceramics.18,19 In fact, in Guarani, an indigenous South American language, genipap means “fruit used to paint”. On the other hand, the ripe genipap fruits are used to manufacture jams and liqueurs.20 There is no information in the literature to justify the formation of the blue pigment only in unripe fruits. Moreover, the investigation of phenolic compound composition and determination of iridoid contents of genipap have never been reported before. Considering these facts, the objective of the present study was to evaluate the influence of the ripeness stage of genipap fruits on their iridoid and phenolic compound composition by high-performance liquid chromatography connected in series to a diode array detector and mass spectrometer.
Figure 1. Basic iridoid structures: (A) simple; (B) glycosylated; (C) secoiridoid.
glycosylated, mainly on the hydroxyl at C-1 or C-11; or (C) secoiridoids, which originate from cleavage of the cyclopentane ring between C-7 and C-8, and bisiridoids, which result from dimerization of iridoids and secoiridoids.2,3 The iridoids can also be esterified with acids, the most common forms being those derived from benzoic and cinnamic acids, and this reaction occurs mainly at positions 6, 8, and 10.1,4 A wide range of biological activities have been associated with iridoids, such as anti-inflammatory activity,3 improvement of short-term memory capacity,5 and activity against gastritis.6 Iridoids can be also used as chemotaxonomic markers for plant classification.7 In addition, some of these compounds have the ability to react with amino acids or proteins to form pigments.8 Genipin, 13, stands out among the iridoids with respect to this property, because it produces a “blue pigment” from its reaction with primary amine sources (Figure 2).9−13 South Korean and Japanese food industries use commercial colorants containing the blue pigment, obtained from Gardenia jasminoides, in products such as sweets, ice creams, condiments, liqueurs, and bakery products.8,12 © 2014 American Chemical Society
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MATERIALS AND METHODS
Standards and Reagents. The genipin and 5-caffeoylquinic acid standards were acquired from Sigma-Aldrich (St. Louis, MO, USA), both with purity of 99% as determined by HPLC-DAD. HPLC grade methanol and acetonitrile were acquired from J. T. Baker (Phillipsburg, NJ, USA), and the analytical grade reagents were bought from Synth (São Paulo, Brazil). Ultrapure water was obtained Received: Revised: Accepted: Published: 10800
July 15, 2014 October 16, 2014 October 17, 2014 October 17, 2014 dx.doi.org/10.1021/jf503378k | J. Agric. Food Chem. 2014, 62, 10800−10808
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Figure 2. Formation reaction for the blue pigment and its appearance in genipap fruit (compounds numbered according to retention time given in Table 2).
Figure 3. Photographs of genipap fruit at two ripening stages: unripe (left) and ripe (right). from a Milli-Q system (Billerica, MA, USA), and the Folin−Ciocalteu reagent was acquired from Dinâmica (São Paulo, Brazil). The samples and standards were filtered through 0.22 μm Millipore membranes. Materials. The genipap fruits (Figure 3) were harvested from a tree situated inside the University of Campinas (UNICAMP) campus, located in the city of Campinas (São Paulo State, Brazil), corresponding to the following coordinates: latitude 22°49′88″, longitude 47°04′21″, and altitude of 606 m. The plant material was authenticated by Professor Jorge Yoshio Tamashiro; Department of Plant Biology at the Institute of Biology, UNICAMP, and a voucher specimen was deposited in the UNICAMP Herbarium (UEC 11713). Three batches of fruits were harvested, with an interval of 10 days between each harvest. All harvests occurred in the months of January and February 2013. The genipap fruits were manually washed and peeled. The portions of mesocarp and endocarp were separated and frozen immediately in liquid nitrogen. Each sample was then freezedried (Liobrás, São Paulo, Brazil) and ground in a domestic food processor. The three batches of each sample were mixed together to form a composite sample and vacuum packed in polyethylene bags. The samples were stored in a freezer at −36 °C until analysis. The moisture of the genipap fruit samples was determined in triplicate according to an AOAC method.21 The samples exhibited the following moisture contents: unripe endocarp, 85.7 ± 0.1 g/100 g; unripe mesocarp, 80.9 ± 0.1 g/100 g; ripe endocarp, 70.2 ± 0.1 g/100 g; ripe mesocarp, 75.3 ± 0.1 g/100 g. Differentiation between Unripe and Ripe Fruits. The ripeness of the fruits was determined by firmness and peel color analysis. These techniques were chosen for being minimally invasive, because tissue damage can both release the enzyme and expose the interior of the unripe fruits to oxygen, which leads to the formation of the blue pigment. All of the fruits used in this study were submitted to firmness and color analysis. The firmness was evaluated by determining the maximum force of penetration (MFP) using a TA-XT Plus texturometer (Stable Micro
Systems Ltd., Surrey, UK) equipped with a P/2N needle probe. The MFP was determined on two opposite faces in the equatorial region of each fruit. The analysis was performed under the following conditions: pretest speed, 5 mm/s; test speed, 2 mm/s; post-test speed, 10 mm/s; and penetration depth, 10 mm. The results were expressed in newtons (N). Fruits with MFP values >10 N were considered unripe, and fruits with MFP values 0.05). L*, a*, and b* are CIELAB color system parameters. a
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Figure 4. HPLC-DAD chromatograms of the iridoids and phenolic compounds of genipap fruits in two ripening stages: (A) unripe endocarp; (B) unripe mesocarp; (C) ripe endocarp; (D) ripe mesocarp. Peak identification is presented in Table 2.
loss of the whole aglycone (212 Da) is characteristic of the presence of an OH group linked to C-8, indicating that the aglycone under question was geniposidic acid, 3.23 For peak 10, the MS3 from the m/z at 323 resulted in fragments with 179 and 161 Da, which correspond to ionized and dehydrated caffeic acid, respectively. For peak 11, the MS3 from the ion with 307 Da resulted in fragments at m/z 217 and 145, which correspond to the sugar moiety and ionized p-coumaric acid, respectively. Thus, compounds 10 and 11 were tentatively identified as caffeoyl geniposidic acid and p-coumaroyl geniposidic acid, respectively. Up to now, the presence of iridoid glycosides esterified with acids in genipap fruits has never been reported in the literature. Peak 12 presented the [M − H]− at m/z 549 and MS/MS fragments at m/z 355 [M − H − 194]− and at m/z 337 [M − H − 194 − 18]−, relative to the loss of one dehydrated and one nondehydrated aglycone, respectively. Considering the above discussion, this fragmentation indicates the absence of an OH group on C-8 or on the carbon directly bound to it.23 Thus, the aglycone present in compound 12 was tentatively identified as that from gardoside, 1. The MS3 of the ion at m/z 355 produced, in addition to the ion at m/z 337 (loss of H2O), an ion at m/z 193, which corresponded to ionized ferulic acid. Thus, compound 12 was tentatively identified as feruloyl gardoside, which was also found in gardenia fruits.31 Peak 13 was identified as genipin, because it showed the same retention time and UV and mass spectra characteristics as
used in this study did not allow the two compounds to be differentiated. Therefore, it was not possible to determine which of the two peaks corresponded to each isomer. Peaks 7 and 9 also formed adducts with the formate ion ([M − H + HCOO]−). Using MS/MS, the [M − H]− fragments at m/z 549 and 387 were observed for peaks 7 and 9, respectively. On the MS3 of the deprotonated molecules of both compounds, the major fragment at m/z 225 corresponded to the aglycone genipin after the loss of one (peak 9) or two (peak 7) hexose units. From the fragmentation pattern, peaks 7 and 9 were tentatively identified as genipin-1-β-D-gentiobioside and geniposide, respectively, on the basis of the similarity of their characteristics with the same compounds found in Gardenia jasminoides Ellis.23 These compounds are the major iridoids in gardenia fruits.26 Peak 8 was identified as 5-caffeoylquinic acid on the basis of the same retention time and UV and mass spectra characteristics as an authentic standard of 5-caffeoylquinic acid. The [M − H]− was detected at m/z 353, and the MS/MS fragments showed 191 and 179 Da due to, respectively, the loss of caffeoyl and quinoyl groups. This is the first study to report the presence of chlorogenic acids in Genipa americana L. Peaks 10 and 11 presented [M − H]− at m/z 535 and at m/z 519, respectively. Using MS/MS, both compounds presented a common loss of 212 Da. This loss could correspond either to the aglycone of gardoside, 1, or geniposidic acid, 3. Considering the same principle used to differentiate compounds 1 and 3, the 10803
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10804
19.8 20.4
22.5
23.9 28.7
29.2
gardenoside DAME or SME
genipin-1-β-Dgentiobioside 5-caffeoylquinic acid
geniposide caffeoylgeniposidic acid
p-coumaroylgeniposidic acid feruloyl gardoside
genipin 3,5-dicaffeoylquinic acid
p-coumaroylgenipin gentiobioside
feruloylgenipin gentiobioside
4,5-dicaffeoylquinic acid
not identified
5 6
7
9 10
11
12
13 14
15
16
17
18
37.9
29.9
29.7
22.9
18.5
17.4
13.9 14.5
302 (sh), 328 276
296(sh), 328
300 (sh), 328 241 298 (sh), 328 294 (sh), 313 298 (sh), 328 238 302 (sh), 328 296(sh), 313
241
241 241
241
236
235
235
λmax (nm)c
nd
515
725
695
225 515
549
519
nd 535
353
nd
nd nd
nd
373
391
373
nd
nd
nd
nd
nd nd
nd
nd
433 nd
nd
595
449 449
449
nd
nd
nde
[M − H]− [M − H + HCOO]− (m/z) (m/z)
549→225
403→241, 223 403→241, 139
403→241, 223, 139
211→167, 149, 123
229→185, 167, 149
211→193, 149, 123
MS3 (m/z)d
353 [M − H − 162]−, 335 [M − H − 162 − 18]−, 173 [M − H − 162 − 162 − 18]−
693, 499 [M − H − 226]−, 397 [M − H − 226 − 102]−, 337 [M − H − 226 − 102 − 60]−, 295, 265, 235
663, 469 [M − H − 226]−, 367 [M − H − 226 − 102]−, 307 [M − H − 226 − 102 − 60]−, 265, 235, 205
307 [M − H − 212]−, 187, 163 [M − H − 212 − 144]−, 145 [M − H − 212 − 162]− 355 [M − H − 194]−, 337 [M − H − 194 − 18]−, 217, 193 [M − H − 212 − 144]−, 175 [M − H − 212 − 162]− 207 [M − H − 18]−, 123 [M − H − 102]−, 101 353 [M − H − 162]−, 191 [M − H − 162 − 162]−
353→191, 179 [M − H − 162 − 174]−, 173 [M − H − 162 − 162 − 18]− 469→367, 307, 265, 235, 163 [M − H − 226 − 162 − 144]−, 145 [M − H − 226 − 180]− 499→397, 295, 265, 235, 193 [M − H − 226 − 162 − 144]−, 175 [M − H − 226 − 180]− 353→191, 179, 173, 135 [M − H − 162 − 174 − 44]−
355→337, 217, 193
307→217, 145
387 [M − H]−, 225 [M − H − 162]−, 123 [M − H − 162 − 102]− 387→225, 123 491 [M − H − 44]−, 323 [M − H − 212]−, 161 [M − H − 212 − 162]− 323→179 [M − H − 212 − 144]−, 161
191 [M − H − 162]−, 179 [M − H − 174]−, 173 [M − H − 162 − 18]− 191→173, 127, 109
211 [M − H − 162]−, 193 [M − H − 162 − 18]−, 149 [M − H − 162 − 44 − 18]−, 123 [M − H − 162 − 44 − 44]− 229 [M − H − 162]−, 185 [M − H − 162 − 44]−, 167 [M − H− 162 − 44 − 18]−, 149 [M − H − 162 − 44 − 18 − 18]− 211 [M − H − 162]−, 167 [M − H − 162 − 44]−, 149 [M − H − 162 − 44 − 18]−, 123 [M − H − 162 − 44 − 44]− 403 [M − H]−, 241 [M − H − 162]−, 223 [M − H − 162 − 18]−, 139 [M − H − 162 − 102]− 403 [M − H]−, 241 [M − H − 162]−, 223 [M − H − 162 − 18]− 403 [M − H]−, 241 [M − H − 162]−, 223 [M − H − 162 − 18]−, 139 [M − H − 162 − 102]− 549 [M − H]−, 225 [M − H − 162 − 162]−
MS/MS (m/z)d
Numbered according to the chromatograms in Figure 4. bRetention time on the C18 column. cSolvent: linear gradient of water and acetonitrile, both containing 0.1% formic acid. dIons in bold type represent the major fragment. end, not detected; fDAME, deacetylasperulosidic methyl ester; SME, scandoside methyl ester
a
13.4
DAME or SMEf
4
8
12.9
geniposidic acid
3
12.0
shanzhiside
2
11.2
tR (min)b
gardoside
compound
1
peaka
Table 2. Chromatographic and Spectroscopic Characteristics of the Iridoids and Phenolic Compounds Obtained from Genipap
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Figure 5. Proposed chemical structures for the compounds found in genipap fruits. The compounds are numbered according to their elution order from the C18 column (Table 2).
Table 3. Iridoid and Phenolic Compound Contents in the Endocarp and Mesocarp of Ripe and Unripe Genipap Fruitsa unripe fruit peak
compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
gardosideb shanzhisideb geniposidic acidb DAME or SMEb gardenosideb DAME or SMEb genipin-1-β-D-gentiobiosideb 5-caffeoylquinic acidd geniposideb caffeoylgeniposidic acidd p-coumaroylgeniposidic acidd feruloyl gardosided genipinb 3,5-dicaffeoylquinic acidd p-coumaroylgenipin gentiobiosided feruloylgenipin gentiobiosided 4,5-dicaffeoylquinic acidd not identifiedb
total iridoids total phenolic compounds
endocarp ndc 4.34 19.86 0.80 7.05 1.37 nd 5.59 107.11 1.34 1.43 0.75 3.40 nd nd nd nd nd
± ± ± ± ±
0.03 0.09 0.02 0.13 0.05
± ± ± ± ± ±
0.06 0.74 0.02 0.02 0.01 0.11
147.45 ± 0.88 5.59 ± 0.06
ripe fruit mesocarp 0.76 4.23 2.54 nd nd nd nd 1.72 117.99 1.44 nd 1.12 nd 1.23 nd nd 1.22 nd
± 0.03 ± 0.04 ± 0.05
± 0.03 ± 0.93 ± 0.02 ± 0.04 ± 0.03
± 0.04
128.07 ± 1.09 4.17 ± 0.07
endocarp nd 1.64 0.19 0.11 0.90 0.09 2.45 nd 0.29 0.07 0.12 0.07 nd nd 0.38 0.10 n.d 1.76
± ± ± ± ± ±
0.06 0.01 0.00 0.03 0.00 0.13
± ± ± ±
0.01 0.00 0.00 0.00
± 0.00 ± 0.01 ± 0.05
6.43 ± 0.22 0.00 ± 0.00
mesocarp nd nd nd nd nd nd nd nd nd 0.06 0.12 0.08 nd nd nd nd nd 1.07
± 0.00 ± 0.00 ± 0.00
± 0.00
0.25 ± 0.00 0.00 ± 0.00
Values of mean ± standard deviation are expressed in mg/g of freeze-dried sample; analyses were carried out in triplicate from the sample pool composed of three batches. bQuantitated as genipin equivalents. cnd, not identified. dQuantitated as 5-caffeoylquinic acid equivalents.
a
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iridoid in the ripe endocarp and was not detected in unripe genipap fruits. This indicates that ripening favors the addition of a second hexoside group to the geniposide, 9. According to the biosynthetic pathway of iridoids, geniposidic acid, 3, is the precursor of geniposide, 9, that further gives rise to gardenoside, 5.7 These three compounds are the major ones found in the unripe genipap endocarp and are among the main compounds of the unripe mesocarp, but their contents indicate that in the unripe genipap fruits, the formation of geniposide, 9, is the favored route. Another difference observed between both ripeness stages was the absence of chlorogenic acids in the ripe genipap fruits. In addition, the ripe genipap fruits presented a 90% reduction in the iridoid contents, especially geniposide, 9, in relation to the green fruits. This behavior was also observed in Gardenia jasminoides fruits, in which the geniposide, 9, contents decreased throughout the fruit-ripening process.39,40 This fact can be explained by the highest expression levels of the enzyme responsible for iridoid glycosylation in the earlier stages of Gardenia jasminoides ripening.41 This phenomenon was attributed to the fact that the iridoids as well as the phenolic acids act as defense compounds against herbivores, and their synthesis demands precursors of low molecular mass and low enzymatic activity, making their synthesis energetically favorable in the initial developmental stages.42−44 Blue Color and Genipap Ripeness Stage. Although the endocarp of the unripe fruits showed a low concentration of genipin, 13 (3.40 mg/g of the freeze-dried sample), small lines of blue color were visually observed, most probably due to formation of the blue pigment as the result of a reaction, shown in Figure 2, between amino acids and free genipin, 13, present in endocarp. In addition, we verified that extracts from unripe genipap fruit kept at room temperature for a few hours resulted in increased intensity of the genipin, 13, peak in the endocarp extract or its appearance in the mesocarp extract (data not shown). In addition, reduction of the geniposide, 9, concentration and formation of a new peak (tR = 21.1 min, [M − H]− at m/z 387 and MS/MS fragments at m/z 285 [M − H − 102]−, 207 [M − H− 162 − 18]−, 179 [M − H − 162 − 18 − 28]−, and 179 [M − H − 162 − 18 − 28 − 18]−) were also observed. This new peak may be an intermediate in the reaction shown in Figure 2. These facts indicate the presence of β-glucosidase in the fruit, which was possibly released by tissue rupture. As shown in Figure 2, the enzyme hydrolyzes the glycosidic bond of geniposide, 9, furnishing genipin, 13, that reacts with primary amine sources present in the matrix. The drastic reduction of geniposide, 9, contents and the absence of genipin, 13, in the ripe fruit may explain the nonoccurrence and/or formation of the blue pigment in the ripe fruits. The present study was the first to relate the iridoid and phenolic compound compositions to both ripening stage and part of the fruit (mesocarp and endocarp). Our results show that the stage of ripeness influences both the iridoid and phenolic compound profiles. The unripe genipap fruits were shown to be a very good source of geniposide, 9, which can be used to produce genipin, 13, and subsequently the blue pigment for use by the natural dye industry. In addition, the fact that geniposide, 9, was almost nonexistent in the ripe genipap fruits could explain the nonformation of the blue pigment in this ripeness stage.
the authentic genipin standard. This compound presented the [M − H]− at m/z 225 and the following MS/MS fragments: m/ z 207 relative to the loss of a water molecule; m/z 123 generated by the loss of a methyl 3-oxopropanoate group (102 Da); and m/z 101, corresponding to the ionized methyl 3oxopropanoate group. Peaks 14 and 17 both presented the same [M − H]− at m/z 515 and a major MS/MS fragment at m/z 353, which is characteristic of dicaffeoylquinic acids. The MS3 fragmentations of the ion at m/z 353 of both compounds were very similar, differing only with respect to the intensity of the major peak. Compound 14 presented a major fragment at m/z 191, resulting from the loss of a caffeoyl group (162 Da), whereas for compound 17 the major fragment was observed at m/z 173, representing a dehydrated quinic acid. The fragmentation pattern of isomers of chlorogenic acids depends on the position of attachment of the caffeoyl group to quinic acid.28 Thus, peaks 14 and 17 were tentatively identified as 3,5dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, respectively. The chlorogenic acids were also found in gardenia fruits.27 Peak 15 showed the [M − H]− at m/z 695 and a major MS/ MS fragment at m/z 469, due to the loss of a genipin molecule (226 Da). The MS3 of this ion showed fragments with 367, 307, 265, and 235 Da, characteristic of the fragmentation of two hexose molecules, and also fragments at m/z 163 and 145, which correspond to ionized and dehydrated p-coumaric acid, respectively. Taking into account all of the information provided, peak 15 was tentatively identified as p-coumaroyl genipin gentiobioside, which was also found in gardenia fruits and in a formulation of a Chinese medicine based on gardenia fruits and fermented soybean.29,36 Peak 16 was tentatively identified as feruloyl genipin gentiobioside, considering the same MS fragmentation mechanism and a difference of 30 Da as compared to pcoumaroylgenipin gentiobioside, 15. However, in this case ions at m/z 193 and 175 were obtained on the MS3, representing ionized and dehydrated ferulic acid, respectively. Feruloyl genipin gentiobioside, 16, has been previously also detected in gardenia fruits.31,37 Peak 18 was not identified, because this compound was detected only on the UV spectrum, but was not ionized under the conditions used in this study, neither in the positive nor in the negative mode. Its late elution on the reversed-phase column indicated low polarity in relation to the other compounds. This was the major compound found in the mesocarp of ripe fruits. Contents of Iridoids and Phenolic Compounds. Because genipap (Genipa americana L.) and Gardenia jasminoides fruits belong to the same family, it is not a surprise that their qualitative compositions of iridoids and chlorogenic acids are very similar. As in gardenia, geniposide, 9, was the major iridoid in the unripe genipap fruits (Table 3), corresponding to 72 and 92% of the total iridoids in the endocarp and mesocarp, respectively. Furthermore, the geniposide, 9, contents in the endocarp and mesocarp of unripe genipap fruits (107.11 and 117.99 mg/g freeze-dried sample, respectively) were higher than those reported for gardenia fruits from different varieties and origins (43.19−95.51 mg/g dry sample).16,38 In addition, the unripe genipap also presented higher geniposidic acid, 3 (endocarp and mesocarp), and gardenoside, 5 (endocarp), contents than those found in gardenia fruits (0.80 and 1.08 mg/g dry sample, respectively).26 Genipin-1-β-D-gentiobioside, 7, was the major 10806
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AUTHOR INFORMATION
Corresponding Author
*(A.Z.M.) Phone: +55-19-35212163. Fax: +55-19-35212153. E-mail:
[email protected] or
[email protected]. Funding
We thank the Brazilian funding agencies Coordination of Improvement of Higher Education Personnel (CAPES) and São Paulo Research Foundation (FAPESP, proc. 2013/079148) for their financial support. Notes
The authors declare no competing financial interest.
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