Distribution of Major Chlorogenic Acids and Related Compounds in

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Distribution of major chlorogenic acids and related compounds in Brazilian green and toasted Ilex paraguariensis (maté) leaves Juliana de Paula Lima, Adriana Farah, Benjamin King, Tomas De Paulis, and Peter Robert Martin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00276 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on February 29, 2016

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Journal of Agricultural and Food Chemistry

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Distribution of major chlorogenic acids and related compounds in Brazilian

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green and toasted Ilex paraguariensis (maté) leaves

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Juliana de Paula Lima,a Adriana Farah,a* Benjamin King,a Tomas de Paulis,b Peter R. Martin.b

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a

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Rio de Janeiro, Ilha do Fundão, CCS bloco J, 21941-902, Rio de Janeiro, RJ, Brazil.

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b

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Vanderbilt Psychiatric Hospital, Suite 3068, 1601 23rd Avenue South, Nashville, TN 37212-

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8645

Lab. Química e Bioatividade de Alimentos, Instituto de Nutrição, Universidade Federal do

Psychiatry Department, Vanderbilt University School of Medicine,

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* Corresponding author:

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e-mail: [email protected]; [email protected]

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Phone/fax: 55-21-3938-6449;

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ACS Paragon Plus Environment


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ABSTRACT

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Ilex paraguariensis (maté) is one of the best sources of chlorogenic acids (CGA) in nature.

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When leaves are toasted, some isomers are partly transformed into 1,5-γ-quinolactones

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(CGL). Both CGA and CGL are important contributors to the brew’s flavor and are thought

30

to contribute to human health. In this study, we quantified 9 CGA, 2 CGL, and caffeic acid, in

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20 samples of dried green and toasted maté that are commercially available in Brazil. Total

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CGA content in green maté varied from 8.7 to 13.2 g/100g, dry weight (dw). Caffeic acid

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content varied from 10.8 to 13.5 mg/100g dw, respectively. Content in toasted maté varied

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from 1.5 to 4.6 g/100g and from 1.5 to 7.2 mg/100g dw, respectively. Overall, caffeoylquinic

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acid isomers (CQA) were the most abundant CGA in both green and toasted maté, followed

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by dicaffeoylquinic acids (diCQA), and feruloylquinic acids (FQA). These classes accounted

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for 58.5%, 40.0% and 1.5% of CGA, respectively, in green maté, and 76.3%, 20.7% and

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3.0%, respectively, in toasted maté. Average contents of 3-caffeoylquinolactone (3-CQL) and

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4-caffeoylquinolactone (4-CQL) in commercial toasted samples were 101.5 mg/100g and 61.8

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mg/100g dw, respectively. These results show that, despite overall losses during the toasting

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process, CGA concentrations are still substantial in toasted leaves, compared to other food

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sources of CGA and phenolic compounds in general. In addition to evaluating commercial

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samples, investigation of changes in CGA profile and formation of 1,5-γ-quinolactones was

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performed in experimental maté toasting.

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KEYWORDS: Maté, yerba mate, chlorogenic acids, 1,5-γ-quinolactones, quinides, Ilex

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paraguariensis, phenolic compounds

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INTRODUCTION

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Originated in the subtropical region of South America, maté (Ilex paraguariensis) is a

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plant species naturally grown and widely cultivated in Brazil (in the states of Paraná, Rio

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Grande do Sul and Santa Catarina), Argentina (in the Northeast of Corrientes and Misiones

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provinces), and Paraguay.1

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Currently, Brazil is the largest world producer of maté (600,000 tons/year).2 Its

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consumption is widespread in the South and Southern regions of the country, given the natural

56

incidence of the herb in the region, as well as for cultural reasons. The indigenous Quechua

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and Guarani nations kept the habit of drinking unfiltered infusions from these leaves and to

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this day, the product obtained from maté dry leaves, also named “erva-mate” or “yerba mate”,

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is still used for the preparation of several types of infusions, for example, hot chimarrão and

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cold terere, both from green leaves, and hot or cold maté tea, from toasted leaves.3 Rio

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Grande do Sul is the state with highest consumption of maté as green “chimarrão” (70,000

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tons/year), while Rio de Janeiro is the largest consumer of

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tons/year).4 Practically all the remaining amount produced is exported. The main destinations

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are Uruguay and Chile and the remaining percentage goes to the US, Europe and Asia, in the

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form of whole or ground dried leaves, or extracts to be used in different phytopharmaceutical

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preparations.5

toasted “maté tea” (1,500

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In addition to the consumption inherent to traditional cultural habits, the consumption

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of maté has increased considerably in the last decade, due to the knowledge of the potential

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health benefits related to its antioxidant 6-9 and anti-inflammatory 10-12 effects, studied in vitro

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and in animals. Maté extracts have exhibited potent in vitro inhibition of oxidative stress

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caused by reactive oxygen species (ROS)1 and were able to protect DNA from in vitro

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oxidation and low-density lipoprotein (LDL) from lipoperoxidation.13 Maté was also able to

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reduce acute lung inflammation and oxidative damage in mice exposed to cigarette smoke.12,14

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The regular consumption of maté tea significantly contributes to the overall antioxidants

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intake in the South region of Brazil, where maté alone is responsible for about 43% of the

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dietary antioxidant capacity, 15 as well as in other consumer countries. Several additional potential effects have been reported for mate, both in vivo and in

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including

hepatoprotective,16,17,18

neuroprotective,19

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vitro,

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antimicrobial,21,22 anti-obesity11,23, and hypocolesterolemic.24,25

diuretic,

antirheumatic,1,20

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The pharmacological effects of maté have been attributed to its bioactive compounds,

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namely the methylxantines caffeine and theobromine (1-2% and 0.5-0.9% of the dried green

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leaves, respectively)

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phenolic antioxidant compounds in maté

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average, 10% of dried green leaves.32 Such high content makes it one of the main sources of

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CGA in nature. Saponins and minor flavonols like rutin, quercetin and kaempferol and other

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minor bioactive compounds are also present.7, 20,33,34

26,27

and especially to chlorogenic acids (CGA) 25,30,31

17,18,20,24,29

, the main

(Figure 1- A and B), accounting for, on

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The green maté used for preparation of hot chimarrão and cold tererê is industrially

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obtained by scorching, crushing and drying of leaves and stems.3 For the preparation of maté

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tea, green leaves are toasted at variable temperatures and times prior to being crushed and

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dried. While many green maté components remain unchanged during toasting,32,35 the high

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temperatures applied during industrial processing of maté (250 ºC – 550 ºC- direct fire, for 2

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- 4 min), decrease its antioxidant activity 36 and could potentially affect its pharmacological

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properties. This is probably caused mainly by degradation of CGA compounds.35 However,

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there are no studies investigating changes in CGA content and distribution during maté

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toasting. Moreover, most studies investigating CGA content in maté have quantified mainly

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CQA and a few CQA and diCQA, expressing the total content as equivalents of CGA.

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Additionally, the presence of bioactive cinnamoyl-1,5-γ-quinolactones or quinides

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(CGL) produced by dehydration of CGA and formation of an intramolecular ester bond

99

between positions 1 and 5 of the quinic acid moiety

37

(Figure 1C) has been observed in

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toasted maté,32 but to our knowledge, their formation and degradation during toasting has not

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been reported. [FIGURE 1]

102 103 104

In addition to their antioxidant effects, in the last decade, these lactones have received

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special attention due to their potential effects on brain function, such as inhibition of

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adenosine transport, affinity for the µ-opioid receptor observed in mice, 38 and antinociceptive

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and glycemy control effects in rats.

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flavor, 40,41,42,43 and possibly confer similar characteristic to maté tea.44

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Like caffeic acid, CGL contribute bitterness to coffee

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In view of the limited reported data, in this study we evaluated the content and

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distribution of CGA and CGL in Brazilian commercial samples of green and toasted leaves of

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Ilex paraguariensis (maté). Additionally, we investigated the changes in CGA profile and

112

formation of CGL in experimental toasting and compared data with those from commercial

113

samples.

114 115

MATERIALS AND METHODS

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Ilex paraguariensis samples: Eight dried green and twelve dried toasted premium maté (Ilex

117

paraguariensis) samples were obtained from reliable commercial sources and producers in the

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South (Santa Catarina, Rio Grande do Sul and Parana states) and Southern (Rio de Janeiro

119

and São Paulo states) regions of Brazil. Dried green maté leaves used in the toasting

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experiments were generously donated by the Brazilian Agricultural Research Corporation in

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Rio Grande do Sul (EMBRAPA, RS, Brazil).

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Toasting: Two grams of unground green maté leaves were placed into separate watch glasses.

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Leaves were heated in an oven (LF0910, Jung, Santa Catarina, Brazil) for 1, 2 and 4 min at

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250 °C (oven chamber temperature). Each leaf was turned over with tweezers halfway

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through heating. Toasting was performed in duplicate.

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Water content: In order to express the content of CGA and CGL on dry weight basis, the

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water content of the dried green and toasted leaves was determined with a MX-50 moisture

130

analyzer (A&D Company, Japan).

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Luminosity: The degrees of luminosity were evaluated using a ColorGap 1A colorimeter

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(Leogap Ind., São Paulo, Brazil).

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Chlorogenic acids extraction: The dried leaves of both green and toasted maté were

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macerated using mortar and pestle and ground to pass a 0.50 mm sieve. Samples were

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extracted with aqueous methanol 40% at room temperature (25°C) for 20 min, as reported by

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Farah et al.

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< 5% for 5-CQA, 4-CQA, 3-CQA, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA, caffeic acid, 3-CQL

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and 4-CQL and between 5-10% for 3-FQA, 4-FQA and 5-FQA. Samples were then analyzed

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in duplicate. Precipitation of proteins and other high molecular weight compounds was

142

performed with Carrez solutions. 37

37

Extraction variation coefficient calculated from sextuplicate of extraction was

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Standards: 5- Caffeoylquinic acid (5-CQA) was purchased from Sigma-Aldrich (St Louis,

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MO). A mixture of 3-CQA, 4-CQA and 5-CQA was prepared from 5-CQA, using the

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isomerization method of Trugo & Macrae.45 The lactones 3-CQL and 4-CQL were

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synthesized using the low temperature modification method as described.46,47 For diCQA, a

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mixture of 3,4-, 3,5-,and 4,5-diCQA from Carl Roth (Karlsruhe, Germany) was used. The

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present work used IUPAC numbering system for CGA identification.48 Although under

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IUPAC rules the order of carbon atoms in the quinolactones is reverse to that of the quinic

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acid,48 to avoid confusion, here we have used the same numbering of the carbon atoms of the

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lactones as for their CGA precursors.

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Chromatographic analysis: Chlorogenic acids and lactones were analyzed by a reverse-

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phase high performance liquid chromatography-diode array detector (HPLC-DAD) system. 49

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Chromatographic separations were achieved using a Magic C30 HPLC column (150 x 2 mm,

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5 µm, 100 Å, Michrom Bioresources, Inc., Auburn, CA, USA) maintained at a constant

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temperature of 40 ºC. The LC two-phase mobile system consisted of 0.3% aqueous formic

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acid (eluent A) and methanol (eluent B). The gradient was programmed to operate with a flow

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rate of 1.0 mL/min, and DAD was set to 325 nm. After preliminary identification of maté

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peaks with LC-MS as described by Marques and Farah,32 because the chromatographic

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profiles of maté samples were similar, the identification of CGA and CGL was performed by

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comparison of target compounds’ retention times with those of the respective standards.

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Additionally, UV spectra were used for peaks confirmation. The quantification of all CGA

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and CGL was performed using the area of 5-CQA standard combined with the molar

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extinction coefficients of the CGA and direct CGL precursors as reported by Ruback50 and

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explained in Farah et al.37 The quantification limit for CGA under these conditions was 0.003

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µg/mL (peak area equivalent to three times the area of baseline noise). Results were presented

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as mean ± standard deviation (SD).

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Statistical analysis: The chromatographic results were statistically tested for differences

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among individual commercial and lab toasted samples by one way ANOVA. Comparison

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between average content of total CGA in green and toasted samples was performed by non-

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paired T-test. For this, GraphPad Prism® software, version 5.0 (San Diego, CA, USA) was

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used and results were considered at 95% confidence level.

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RESULTS AND DISCUSSION

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Chlorogenic acids in green I. paraguariensis leaves: Nine CGA compounds and caffeic

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acid (CA) were quantiﬁed in all untoasted maté samples: 3-CQA, 4-CQA, 5-CQA, 3-FQA, 4-

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FQA, 5-FQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA (Figure 2A). The results are presented

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in Table 1 and Figure 3.

183 184

[Figure 2, Table 1, Figure 3]

185 186

Total CGA content varied from 8.7 to 13.2 g/100 g dry weight (dw), with average of

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11.5 ± 1.5 g/100 g dw (Table 1). Caffeic acid contents varied from 10.8 to 13.5 mg/100g dw.

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As with any plant food, maté would show natural variations in physical and chemical

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characteristics due to the influence of genetic aspects, age of tree and leaves, cultivation

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system, climate and soil, harvesting time, processing and storage.

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losses have occurred in some of the samples during primary processing/drying. No significant

3

It is also possible that

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differences were observed among average total CGA contents of green maté samples from

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distinct origins (Santa Catarina, Paraná, and Rio Grande do Sul).

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Generally, the total CGA contents observed in these samples are consistent with

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previous data for green maté leaves. 20,26,32 However, comparisons are difficult to make, since

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as previously mentioned, earlier studies that evaluated the phenolic composition of Ilex

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paraguariensis leaves

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diCQA, expressing the total content (from 0.9 to 10.7 g/100g) as equivalents of CGA.

6,44,51,52

identified and quantified mostly only CQA or CQA and

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CQA, followed by di-CQA, were the most abundant CGA compounds in all green

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maté samples, in agreement with previous findings.26,32 The average content of CQA isomers

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in green maté leaves corresponded to 59% of total CGA; diCQA to 40%; and FQA

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contributed only 1.5% of total CGA (Figure 3). Interestingly, the percentage of diCQA is

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considerably higher in maté comparing to green coffee (15 - 18%),37 another major source of

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CGA in plants. FQA percentage in green coffee (about 5-6%) is also higher comparing to

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maté leaves. No additional major peaks were observed in the chromatograms, except for one

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peak in green maté, between 3,5-diCQA and 4,5-diCQA. In our previous study reporting

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CGA content in medicinal herbs,

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acid and p-coumaric acid were identified in maté. Also, the presence of additional minor CGA

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like diferuloyl quinic acids, caffeoyl-p-coumaroylquinic acids, caffeoyl-feruloylquinic acids,

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caffeoyl-sinapoylquinic acids, tricaffeoyl-quinic acid and dicaffeoyl-feruloylquinic acid and

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similar

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tricaffeoylshikimate and feruloylshikimate) has been previously reported

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minor compounds and in the present study we aimed only at major CGA compounds, except

214

for 4-FQA and 5-FQA, that we were able to quantify.

compounds

like

32

peaks with m/z compatible with caffeoyl-feruloylquinic

shikimates

(caffeoylshikimates,

dicaffeoylshikimates, 29,53

but they were

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Regarding individual CQA isomers, 3-CQA was predominant (average 56% of total

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CQA and 33% of total CGA). The predominance of 3-CQA isomer in green maté leaves,

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followed by 5-CQA and 4-CQA is in agreement with previous findings.26,32

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Regarding diCQA, 3,5-diCQA was the major isomer (about 69% of total diCQA and

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27.6% of total CGA), followed by 4,5-diCQA (23.6% of total di-CQA and 9.3% of total CGA

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) and 3,4-diCQA (7.9% of total di-CQA and 3.1% of total CGA). Among the main FQA, 3-

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FQA was responsible for about 54% of total FQA, followed by 4-FQA and 5-FQA (26.2%

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and 19.7% of total FQA, respectively). The content of caffeic acid in green maté varied from

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10.8 to 13.5 mg/100g. Similar content of caffeic acid (14-20 mg/100g) have been

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reported.44,52 No CGL were identified in green maté samples.

225 226

Chlorogenic acids and cinammoyl-1,5-quinolactones in toasted I.paraguariensis leaves:

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In addition to the nine CGA quantified in commercial dried green maté samples, two

228

cinamoyl-1,5-lactones (CGL), 3-caffeoylquinic-1,5-lactone (3-CQL) and 4-caffeoylquinic-

229

1,5-lactone (4-CQL), were identified and quantified in all toasted maté samples (Figure 2B).

230

Results are presented in Table 2 and Figure 3.

231

[Table 2, Figure 3]

232 233

Total CGA contents varied considerably among the toasted samples, from 1.5 to 4.6

234

g/100g dw, with average content (2.9 g/100g), which is 26% lower than that observed in

235

green samples (Table 2). This was expected because, as previously stated, loss of CGA during

236

heat exposure has been reported for both maté

237

commercial toasted maté samples did not necessarily originate from the green samples

32,36

and coffee.37,49 However, because the

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evaluated in this study, the corresponding losses during toasting could not be determined in

239

this section of the study.

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In addition to the causes described for variations in the composition of green maté,

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differences in toasting conditions were probably the major factor responsible for the large

242

variation in CGA content in these leaves. A positive linear correlation was observed between

243

degree of luminosity and total CGA content (r = 0.93, p < 0.001), showing that samples with

244

higher degree of luminosity (lower toasting degree) contained more total CGA (Figure 4).

245 246

[Figure 4]

247 248

CQA was the most abundant subgroup of CGA compounds in toasted mate samples as

249

in green maté, but with relative increase (76% average contribution to total CGA), followed

250

by di-CQA (21%) and FQA (3%). The increase in percentage of CQA associated with a

251

decrease in diCQA in toasted maté suggest a breakage in the ester bond between caffeic acid

252

and (-) quinic acid of diCQA and formation of CQA and caffeic acid, whose levels varied

253

from 1.5 to 8.1 mg/100g. Samples with lower toasting degree (lighter samples) presented

254

higher caffeic acid content, which is in agreement to the fact that this compound is also heat

255

sensitive. Therefore, it is probably degraded or given another fate as soon as it is formed such

256

as being incorporated to melanoidins from Maillard Reaction, for example, as it happens for

257

coffee, since it has been reported that caffeic and chlorogenic acids are incorporated in coffee

258

melanoidins’ backbone during roasting.55,56 Such melanoidins would be at least partially

259

responsible for the darker color (lower degree of luminosity) of the roasted samples.

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The individual isomer distribution was different in toasted compared to green maté.

261

While 3-CQA was the predominant CQA isomer in green maté, 5-CQA predominated in

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toasted maté, corresponding, on average, to 42% of total CQA and 32% of total CGA.

263

Average content of 3-CQA corresponded to 24% of total CQA in toasted maté and 18% of

264

total CGA. This is an interesting observation in comparison to coffee matrix, where 5-CQA is

265

predominant in unroasted beans and 3-CQA and 4-CQA are rapidly formed by isomerization

266

increasing their content during heat exposure. Following CQA pattern, 5-FQA was the most

267

abundant isomer in toasted maté, and with average contents corresponding to 47% of total

268

FQA, followed by 4-FQA (32%) and 3-FQA (21%).

269

In contrast to green maté samples, 4,5-diCQA was consistently the most prevalent

270

diCQA isomer in toasted maté leaves, corresponding to about 54% of total diCQA, while 3,4-

271

CQA and 3,5-CQA contents corresponded to 16% and 30%, respectively.

272

Regarding the quinolactones, 3-CQL and 4-CQL were only identified in toasted maté,

273

with average contents of 102 ± 31 and 62 ± 33 mg/100g dw, respectively. As in coffee, 3-

274

CQL was the most abundant CGL in all samples (Table 2). These CGL contents in toasted

275

maté leaves are in agreement with our previous report in which mass spectrometry was used

276

for peaks identification (m/z 335).32 Jaiswal et al.53 reported the existence of minor

277

caffeoylshikimic acids that could be confounded with CGL and, according to the authors,

278

shikimic acid esters are not products from the leaf processing but are genuine mate secondary

279

metabolites. In another study 57 the authors reported that these minor shikimates could only be

280

distinguished from CQL by LC-MSn. However, although we did not use LC-MSn in the

281

present study, the fact that we did not find these peaks in green maté and that they were major

282

peaks in toasted maté demonstrate clearly that these peaks are not shikimates. The similarity

283

of CQL contents of maté and coffee is another evidence, since green coffee (Coffea

284

canephora) and green maté often have similar CGA content and in this study the content of

285

lactones was in the same magnitude to that in roasted coffee.37,58,59 Furthermore, since the

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chromatographic column used in the present study was different from the one used in Jaiswal

287

et al.53, it is possible that the referred shikimate had another elution time, being perhaps one of

288

the minor peaks next to 3-CQL. The presence of additional lactones in toasted mate leaves,

289

including diCQL, cannot be ignored since diCQA are major constituents in maté.

290 291

As for the green maté samples, no significant difference was observed when comparing toasted samples from different states.

292 293

Experimental toasting of maté leaves:

294 295

In order to verify the differences between green and toasted maté regarding changes in

296

CGA content and profiles, including formation of CGL, we toasted whole dried green leaves

297

of I. paraguariensis, for 1, 2 and 4 min, using an oven at 250 °C.

298

distribution of CGA and CGL compounds under these conditions are presented in Figure 5.

The contents and

[Figure 5]

299 300 301

Total CGA content in green maté leaves was 8.1 ± 0.1 g/100g. The distribution of

302

CGA subgroups in green maté leaves showed a similar pattern to that of the commercial green

303

maté. CQA was the most abundant subgroup of CGA compounds (54.9%), followed by di-

304

CQA (44%), and FQA (1.5%).

305

After 1 and 2 min toasting, we observed 27.3% and 46.4% decreases in CGA, relative

306

to green leaves, respectively. The longest toasting time (4 min) resulted in 83% decrease in

307

CGA content and was associated to the lowest degree of luminosity, similar to that found in a

308

few commercial samples.

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In samples of maté toasted for 1 and 2 min, CQA content corresponded to about 69%

310

and 73% of total CGA, respectively. A decrease in diCQA contribution to total CGA,

311

compared to green maté, was observed after 1 and 2 min of toasting, corresponding to 28%

312

and 23% respectively. An increase in FQA percentage, on the other hand, was observed,

313

corresponding, to 2.9% and 3.3% contribution, respectively. This suggests that FQA are more

314

resistant to heat comparing to diCQA (and possibly CQA) in agreement with previous results

315

observed in coffee.37,49 After 4 min of toasting, CQA compounds contributed 73% of total

316

CGA, with a decrease in diCQA contribution to 23.6% of total CGA content compared to

317

green maté, suggesting that although CQA are degraded and modified during toasting of maté

318

leaves, they are also formed from diCQA. FQA contributed to 3.4% of total CGA.

319

Regarding individual isomers, 3-CQA was the predominant CQA compound in green

320

maté leaves, responsible for 45% of total CQA and 25% of total CGA, while 5-CQA content

321

was equivalent to 30% and 16%, respectively. After toasting, 5-CQA content increased in the

322

first two minutes of toasting, corresponding, on average, to 46% and 46% of total CQA, and

323

increasing to 47% of total CQA in the 4th minute of toasting. 5-CQA corresponded to 32%,

324

34% and 35% of totals CGA after 1, 2 and 4 min toasting.

325

As in commercial samples, FQA followed the CQA pattern. 3-FQA was the most

326

abundant isomer in green leaves (53% of total FQA). In leaves toasted for 1, 2 and 4 min, its

327

contents decreased about 27%, 52% and 80% comparing to its content in green maté. On the

328

other hand, 5-FQA content increased 98% and 87% during the first 2 min of toasting,

329

thereafter decreasing 24% of its original content in green maté.

330

Among diCQA, 3,5-diCQA was the dominant isomer in green leaves, corresponding

331

to about 55% of total diCQA; 3,4-diCQA corresponded to 13% of total diCQA, and 4,5-

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diCQA to 32%. In leaves toasted for 1, 2 and 4 min, 4,5-diCQA was the most abundant

333

isomer, corresponding to 43%, 43% and 51% of total di-CQA content, respectively.

334

Considering the high contents, special attention should be paid to the potential biological

335

properties of 3,5-diCQA and 4,5-diCQA since diCQA have shown potent antioxidant,60

336

antiinflamatory,60 imunostimulant,61 neuroprotective,62 among other effects in vitro and in

337

animals.

338

Regarding the CGL, although 5-CQA, 3-CQA, and 4-CQA are the three major CGA in

339

maté leaves, only the latter two compounds, having no substituent in the 5-position of the (-)-

340

quinic acid, are able to form a 1,5-γ-quinolactone. Therefore, 3-CQL and 4-CQL were

341

expected to be the major lactones in toasted maté, which was confirmed. CGL seemed to

342

reach their maximum levels approximately 1 min after the start of toasting process. 3-CQL

343

was the most abundant 1,5-γ-quinolactone in all samples, reaching its maximum amount after

344

1 min of toasting (140 mg/100g). 4-CQL showed its maximum amount also after 1 min of

345

toasting (109 mg/100g). The levels of total CQL (249 mg/100g) were lower than maximum

346

levels observed in roasted coffee

347

toasting. The average amount of 3-CQL and 4-CQL after 1 min of toasting represented 1.7%

348

and 1.3% of total content of CGA in green maté leaves, respectively, and 6.9% and 9.8% of

349

the initial mean values of their direct precursors, 3-CQA and 4-CQA, respectively. A similar

350

equilibrium between 3-CQL and 4-CQL, and 3-CQA/4-CQA has been previously observed in

351

coffee.37,49,58,59 The higher levels of 3-CQL as compared to 4-CQL could be explained by the

352

higher amount of the precursor 3-CQA. During lactone formation, 3-CQL is generated also

353

from 4-CQL. When 4-CQL is formed, elimination of a water molecule from the axial chair

354

conformer of the cyclohexane ring of 4-CQA may occur. Because the equatorial conformer is

355

thermodynamically more stable, the equilibrium between the equatorial and the axial chair

37,49

and could have possibly been higher before 1 min

15



Page 16 of 41

356

conformers tends to be shifted until most of 4-CQA is transformed.37,49 This is not as clearly

357

observed here as it is in coffee, since the percentage of 4-CQL formation in relation to its

358

precursor is higher in maté than in coffee. This finding deserves further investigation.

359

After 2 min of toasting, the levels of total lactones decreased 47% when compared to

360

the sample toasted for 1 min. The average amount of 3-CQL (80.8 mg/100g) and 4-CQL (49.5

361

mg/100g) at 2 min of toasting, represented 0.9% and 0.6% of total content of CGA in green

362

maté leaves, respectively and 4% and 4.4% of the initial mean values of their direct

363

precursors. Longer toasting time (4 min) resulted in lower amounts of both CGA and CGL.

364

The amount of total CGA and CGL decreased to 83% and 52%, respectively, of their maximal

365

values.

366

In conclusion, nine CGA and caffeic acid were identified and quantified in green

367

(untoasted) and toasted commercial maté samples. Additionally, the formation of two CGL

368

was investigated in toasted leaves. Total CGA contents in green maté varied from 8.7 to 13.2

369

g/100 g dw, with average of 11.5 ± 1.5 g/100 g dw. Average content in toasted maté samples

370

was about 75% lower than in green samples. Overall, CQA, followed by di-CQA, were the

371

most prevalent and abundant CGA compounds in all green and toasted maté leaves samples.

372

3-CQA, followed by 3,5-diCQA were the predominant isomers in green samples, while in

373

toasted samples, 5-CQA, followed by 4,5-diCQA predominated.

374

Changes in CGA isomers distribution and CGL formation were investigated during

375

maté toasting. This study demonstrated that the formation of CGL is dependent on the

376

toasting time for a given temperature. Among the evaluated toasting times at 250 °C, the

377

maximum amount of lactones was observed after 1 min of toasting, whereas 2 and 4 min

378

toasting yielded lower amounts. Despite favorable structural configurations of CGA, less than

16


Page 17 of 41


379

3% of the total amount of CGA and about 8% of the direct precursors in green maté were

380

converted to lactones. 3-CQL was the major lactone, followed by 4-CQL.

381

The results from the present study show that, despite overall losses during the toasting

382

process, CGA concentrations are still substantial in toasted leaves, comparing to other food

383

sources of phenolic compounds.

384

maté is important, since it may provide basis to explore the intake of these compounds

385

especially where they are intensely consumed as in the South of Brazil, Paraguay, Uruguay

386

and Argentina. The bioavailability and pharmacological effects of these compounds in

387

humans, after both normal and high maté consumption, need to be investigated. Special

388

attention should be paid to the potential biological properties of 3,5-diCQA in green maté and

389

4,5-diCQA in toasted maté and potential for use as extracts or for isolation of compounds.

63,64

The measurement of major CGA contents in Brazilian

390 391

ACKNOWLEDGMENT

392 393

The authors would like to acknowledge the financial support and scholarships of the Brazilian

394

National Research Council (CNPq) and the Research Support Foundation of Rio de Janeiro

395

(FAPERJ).

396 397 398 399

17



Page 18 of 41

400

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654


*FIGURES CAPTIONS

655 656

Figure 1 (A) The main chlorogenic acids monoesters in maté. CQA = caffeoylquinic acids;

657

FQA = feruloylquinic acids (B) The main chlorogenic acids diesters in maté. diCQA =

658

dicaffeoylquinic acids. Esterification also occurs mainly in carbons 3- and 4- of the (-)quinic

659

acid (C) Formation of a cinnamoyl-1,5-γ-quinolactone (CQL) from a chlorogenic acid

660

compound, using 3-caffeoylquinic acid as an example. The authors adopted the IUPAC

661

numbering system for chlorogenic acids. Although under IUPAC rules the numbering system

662

for the lactones is different from that for the acids, in order to avoid confusion, in this study,

663

the authors used for lactones the same numbering of the carbons as for the acid precursors.

664 665

Figure 2. Typical chromatogram and UV spectra of (A) green and (B) toasted maté (Ilex

666

paraguariensis) with major chlorogenic acids, caffeic acid cinnamoyl-1,5-γ-quinolactones. 1.

667

3-caffeoylquinic acid.; 2. 3-feruloylquinic acid; 3. 5-caffeoylquinic acid; 4. 4-caffeoylquinic

668

acid; 5. 5-feruloylquinic acid; 6. 4-feruloylquinic acid; 7. 3,4-dicaffeoylquinic acid; 8. 3,5-

669

dicaffeoylquinic; 9. 4,5-dicaffeoylquinic acid; 10. caffeic acid; 11. 3-caffeoylquinolactone;

670

12. 4-caffeoylquinolactone. The authors adopted the IUPAC numbering system for

671

chlorogenic acids. Although under IUPAC rules the numbering system for the lactones is

672

different from that for the acids, in order to avoid confusion, in this study, the authors used

673

for lactones the same numbering of the carbons as for the acid precursors.

674 675

Figure 3. (A) Average distribution of major caffeoylquinic acids (CQA), dicaffeoylquinic

676

acids (diCQA) and (B) feruloylquinic acids (FQA), caffeic acid (CA) and caffeoylquinic-1,5-

677

lactones (CGL) in Brazilian commercial green (n = 8) and toasted (n = 12) Ilex

27



Page 28 of 41

678

paraguariensis (maté) leaves. Results are means of duplicate of extraction, expressed on dry

679

weight basis (dw), as mean ± standard deviation. CV ≤ 5% for CQA and di-CQA and CV >

680

5% and ≤ 10% for FQA. CV calculated from sextiplicate of extraction. Different letters over

681

columns for the same compound mean statistical difference at a 95% confidence level. The

682

authors adopted the IUPAC numbering system for chlorogenic acids. Although under IUPAC

683

rules the numbering system for the lactones is different from that for the acids, in order to

684

avoid confusion, in this study, the authors used for lactones the same numbering of the

685

carbons as for the acid precursors.

686 687

Figure 4. Correlation between total chlorogenic acids content and degree of luminosity in

688

Brazilian commercial samples of toasted I. paraguariensis (maté) leaves (n=12).

689 690

Figure 5. Distribution of caffeoylquinic acids (CQA), dicaffeoylquinic acids (diCQA) (A);

691

feruloyiquinic acids (FQA), caffeic acid (CA) and caffeoylquinic-1,5-lactones (CGL) (B) in

692

experimental toasting of Ilex paraguariensis (maté) leaves for 1, 2 and 4 min at 250°C.

693

Results are means of duplicate of extraction, from duplicate of toasting, expressed on dry

694

weight basis (dw), as mean ± standard deviation. CV ≤ 5% for CQA and di-CQA and CV >

695

5% and ≤ 10% for FQA. Different letters over columns for the same compound mean

696

statistical difference at a 95% confidence level. The authors adopted the IUPAC numbering

697

system for chlorogenic acids. Although under IUPAC rules the numbering system for the

698

lactones is different from that for the acids, in order to avoid confusion, in this study, the

699

authors used for lactones the same numbering of the carbons as for the acid precursors.

700

When citing other authors, their numbering has been changed for consistency.

28


Page 29 of 41


701

Table 1 - Content of chlorogenic acids (CGA) and caffeic acid in methanolic extracts of Brazilian commercial dried green I.

702

paraguariensis (maté) leaves.*

Green mate samples and origin G1-SC

Moisture (%)

3-CQA

4-CQA

5-CQA

3-FQA

4-FQA

5-FQA

3,4-diCQA

3,5-diCQA

4,5-diCQA

Caffeic acid

Total CGA and caffeic acid

4.8

4451.6 ± 20.7

1057.5 ± 12.8

1749.6 ± 20.2

109.4 ± 8.1

52.9 ± 2.4

39.7 ± 1.1

563.2 ± 14.9

4227.8 ± 20.3

1008.8 ± 30.3

13.5 ± 0.02

13274.0a

G2-SC

4.7

4651.6 ± 77.5

1231.4 ± 31.5

1849.4 ± 10.5

105.2 ± 6.7

51.7 ± 0.2

35.6 ± 1.6

418.6 ± 7.8

3949.4 ± 50.5

922.1 ± 40.5

12.4 ± 0.03

13227.4a

G3-RG

8.9

4230.8 ± 94.2

1072.5 ± 10.6

1895.6 ± 40.1

99.9 ± 3.9

45.4 ± 2.1

37.2 ± 0.2

366.4 ± 2.3

3692.9 ± 40.4

982.0 ± 40.2

11.5 ± 0.05

12434.2a

G4-PR

4.7

4107.2 ± 18.6

1192.9 ± 40.4

1709.4 ± 30.2

96.2 ± 3.3

44.2 ± 1.3

36.2 ± 0.2

289.4 ± 5.5

3508.8 ± 20.6

1116.1 ± 50.3

12.8 ± 0.01

12113.0a

G5-SC

8.4

3822.5 ± 11.2

1151.6 ± 50.5

1799.6 ± 10.6

91.3 ± 5.1

47.7 ± 1.9

34.3 ± 0.8

361.9 ± 10.2

2875.6 ± 20.8

1278.4 ± 20.6

11.4 ± 0.01

11474.2b

G6-SC

4.8

3970.9 ± 16.0

1087.9 ± 12.3

1373.6 ± 20.4

91.1 ± 5.2

45.6 ± 1.7

32.3 ± 0.1

324.4 ± 2.6

3388.9 ± 20.4

1150.6 ± 50.2

13.2 ± 0.03

11478.5b

G7-PR

8.2

2477.1 ± 28.9

1230.6 ± 10.2

1785.0 ± 10.3

79.2 ± 8.3

39.6 ± 1.6

29.7 ± 3.2

329.6 ± 14.2

2894.7 ± 20.1

1017.9 ± 20.2

12.6 ± 0.02

9895.8c

G8-SC

5.3

2703.6 ± 34.0

1412.3 ± 60.8

1815.3 ± 50.4

74.0 ± 6.1

34.9 ± 2.1

26.2 ± 1.9

270.9 ± 8.1

1458.1 ± 70.1

948.9 ± 40.2

10.8 ± 0.04

8755.0c

3801.9 ± 793.8

1179.5 ± 116.6

1747.1 ± 161.4

93.3 ± 12.1

45.2 ± 5.9

33.9 ± 11.8

365.5 ± 92.3

3249.4 ± 862.3

1053.1 ± 119.8

12.2 ± 0.9

11581.5

Mean *

Results are shown as mean of extractions in duplicate ± standard deviation, expressed in mg/100g, on dry weight basis. CQA = caffeoylquinic acid; FQA = feruloylquinic acid; diCQA = dicaffeoylquinic acid. Origin: SC = Santa Catarina; RS = Rio Grande do Sul; PR = Paraná.

29



Page 30 of 41

703

Table 2 - Content of chlorogenic acids (CGA), caffeic acid and cinnamoyl-1,5-lactones (CGL) in methanolic extracts of Brazilian

704

commercial dried toasted I. paraguariensis (maté) leaves.

Toasted mate samples and origin

Moisture (%)

Degree of luminosity

3-CQA

4-CQA

5-CQA

3-FQA

4-FQA

5-FQA

3,4-diCQA

3,5-diCQA

4,5-diCQA

Caffeic acid

3-CQL

4-CQL

Total CGA and related compounds *

T1-SC

4.3

124

822.6 ± 13.4

1159.4 ± 48.4

1257.4 ± 11.2

11.8 ± 1.7

26.6 ± 1.8

44.8 ± 1.4

200.3 ± 10.3

382.0 ± 12.3

738.8 ± 11.3

7.2 ± 0.3

127.7 ± 1.2

98.4 ± 0.3

4877.0a

T2-SC

4.6

120

885.3 ± 15.3

1201.6 ± 37.1

1306.7 ± 32.3

19.6 ± 1.5

24.0 ± 1.2

35.4 ± 2.3

127.4 ± 6.3

288.7 ± 6.0

493.8 ± 22.1

5.0 ± 0.1

123.7 ± 5.7

95.1 ± 0.4

4606.3 b

T3-PR

5.2

119

780.9 ± 18.3

1040.6 ± 50.6

1336.5 ± 14.3

16.3 ± 1.0

20.6 ± 0.8

33.1 ± 2.2

127.1 ± 6.1

251.3 ± 6.7

438.2 ± 11.4

5.1 ± 0.1

141.6 ± 4.3

102.1 ± 0.2

4293.4c

T4-RJ

5.1

119

683.4 ± 20.3

1047.3 ± 43.2

1229.9 ± 10.3

16.1 ± 1.6

21.8 ± 1.4

32.0 ± 2.9

164.1 ± 7.1

281.4 ± 12.0

552.5 ± 17.0

5.9 ± 0.1

83.6 ± 2.4

44.1 ± 0.4

4162.1c

T5-RJ

3.2

100

514.7 ± 10.5

1198.5 ± 1.4

795.7 ± 34.7

18.7 ± 1.9

31.9 ± 1.5

38.4 ± 2.4

102.1 ± 4.5

188.5 ± 7.4

316.0 ± 12.7

2.3 ± 0.11

58.7 ± 2.2

17.4 ± 0.8

3282.9d

T6-SP

2.9

96

599.4 ± 9.4

752.1 ± 10.7

899.4 ± 40.9

17.3 ± 1.4

27.3 ± 1.7

38.3 ± 2.6

101.6 ± 3.4

183.2 ± 2.3

296.2 ± 5.3

5.0 ± 0.2

66.7 ± 2.3

19.0 ± 0.6

3005.5d

T7-SP

3.4

96

525.1 ± 12.6

684.8 ± 10.6

848.7 ± 54.7

21.8 ± 1.6

26.4 ± 1.5

34.5 ± 2.6

98.6 ± 3.3

138.6 ± 2.0

263.0 ± 6.1

2.1 ± 0.1

109.8 ± 2.4

55.3 ± 1.3

2808.3e

T8-RJ

5.2

92

355.0 ± 15.6

581.6 ± 22.3

850.1 ± 32.1

19.0 ± 1.0

24.7 ± 1.0

32.0 ± 2.3

72.1 ± 2.9

178.6 ± 6.4

344.7 ± 5.2

5.4 ± 0.5

137.9 ± 5.5

106.8 ± 1.2

2707.9e

T09-RG

5.3

92

417.6 ± 22.9

567.8 ± 12.3

807.3 ± 34.6

14.2 ± 1.4

23.6 ± 1.2

40.7 ± 2.0

87.4 ± 3.2

144.3 ± 5.0

260.2 ± 3.0

2.9 ± 0.1

103.7 ± 3.0

62.9 ± 1.2

2532.6e

T10-SC

3.3

78

296.6 ± 12.5

411.2 ± 20.1

579.8 ± 22.0

15.9 ± 1.6

22.6 ± 0.6

39.3 ± 0.7

49.4 ± 1.6

124.4 ± 8.3

230.0 ± 4.2

1.5 ± 0.1

90.8 ± 3.7

55.6 ± 1.1

1917.1f

T11-PR

4.8

76

317.1 ± 12.9

446.1 ± 20.6

705.7 ± 30.6

15.6 ± 1.2

27.2 ± 1.0

38.5 ± 1.4

36.1 ± 2.9

58.3 ± 2.4

92.4 ± 3.8

2.0 ± 0.1

120.1 ± 5.2

63.3 ± 1.0

1922.4f

T12-RG

4.2

72

271.3 ± 24.1

371.5 ± 17.5

537.3 ± 10.1

13.8 ± 0.9

25.2 ± 0.3

39.6 ± 1.5

53.1 ± 2.7

80.0 ± 2.4

137.1 ± 7.1

2.4 ± 0.1

53.9 ± 1.3

22.3 ± 1.5

1597.5 g

539.1 ± 215.8

788.5 ± 322.4

929.5 ± 282.6

16.7 ± 2.8

25.2 ± 3.0

37.2 ± 3.9

101.6 ± 48.0

191.5 ± 94.1

346.9 ± 182.4

3.9 ± 1.8

101.5 ± 30.5

61.8 ± 32.8

3143.6

Mean

*Results are shown as mean of extractions in duplicate ± standard deviation, expressed in mg/100g, on dry weight basis. CQA = caffeoylquinic acid; FQA = feruloylquinic acid; diCQA = dicaffeoylquinic acid; CQL = caffeoylquinic-1,5-lactone. Origin: SC = Santa Catarina; PR = Paraná; RG = Rio Grande do Sul; RJ = Rio de Janeiro; SP = São Paulo. Total CGA and related compounds = CGA + caffeic acid + CQL.

30


Page 31 of 41


FIGURE 1 1 2

(A) OH

R O O

6

HO2C

3 4 5 6

2

OH RR= =OH OH RR= =OCH OCH 3 3 R=H

3

1

OH

OH

5-CQA 5-CQA 5-FQA 5-FQA 5-pCoQA

(B) 6 HO2C 1 OH

13 14 15

4

5

5 2

OR3 4 OR2

R1 = CA, R2 = CA, R3 = H R1 = CA, R2 = H, R3 = CA R1 = H, R2 = CA, R3 = CA

3 OR1

7 8 3,4-diCQA 3,5-diCQA 9 4,5-diCQA10 11 12

(C) O O HO

COOH 3 OH

O 4

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

HO

O

OH

5

1

OH

∆

HO

3O

O

H 2O

4

HO

3-CQA

1

OH

5

OH 3-CQL

31



37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

Page 32 of 41

FIGURE 2 (A)

(B)

32


Page 33 of 41

1 2 3


FIGURE 3 (A)

(B) 4

5000

a

4000

150

3000 a

2000 a

1000

mg/100g

mg/100g

Green Maté Toasted Maté

a

b

b

a

100

a

a

50

a b

b

b

b

0

a

a

b

b a

a

a

0 3-CQA

4-CQA

5-CQA

3,4-diCQA

3,5-diCQA

3-FQA

4,5-diCQA

4-FQA

5-FQA

CA

3-CQL

4-CQL

5 6 7 8 9 10 11 12 13 14 15

33



FIGURE 4

Total CGA content (mg/100g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 34 of 41

6000

Total CGA content r = 0.93 4000

2000

0 60

80

100

120

140


34


Page 35 of 41


FIGURE 5

1 2 3 4 5

(A)

(B) Green Maté Toasted Maté (1min) Toasted Maté (2min) Toasted Maté (4min)

3000 150 a

a

a b

b

1000

a

b

2000

c

mg/100g

mg/100g

a

a c

c

b d d

d

a

b

c

b c c

d

d

4-CQA

5-CQA

3,4-diCQA

3,5-diCQA

4,5-diCQA

b

c

b

c c c

d

c c

b

50

b

b

a

0 3-CQA

a

100

a d

a d

d

0

3-FQA

4-FQA

5-FQA

3-CQL

4-CQL

35



1 2 3 4 5

Page 36 of 41

TOC GRAPHIC

6

36


Page 37 of 41


FIGURE 1 (A) OH

R O O

6

4

5

HO2C

2

OH RR= =OH 5-CQA OH 5-CQA RR= =OCH 5-FQA OCH 3 3 5-FQA 5-pCoQA R=H

3

1

OH

OH

(B) 6 HO2C 1 OH

5 2

OR3 4 OR2

R1 = CA, R2 = CA, R3 = H R1 = CA, R2 = H, R3 = CA R1 = H, R2 = CA, R3 = CA

3

3,4-diCQA 3,5-diCQA 4,5-diCQA

OR1

(C) O O HO

3 OH

O 4

HO

O

COOH

OH

5

1

OH



HO

3O

O

H2O

4

HO

3-CQA

OH 3-CQL


5

1

OH


FIGURE 2 (A)

(B)


Page 38 of 41

Page 39 of 41


FIGURE 3 (A) 5000

(B)

a

4000

150

3000 a

2000 a

1000

mg/100g

mg/100g

Green Maté Toasted Maté

a

b

b

a

100

a

a

50

a b

b

b

0

a

a

b b

b a

a

a

0 3-CQA

4-CQA

5-CQA

3,4-diCQA

3,5-diCQA

4,5-diCQA

3-FQA


4-FQA

5-FQA

CA

3-CQL

4-CQL


Total CGA content (mg/100g)

FIGURE 4

6000

Total CGA content r = 0.93

4000

2000

0 60

80

100

120

140



Page 40 of 41

Page 41 of 41


FIGURE 5

(A)

(B)

3000

Green Maté Toasted Maté (1min) Toasted Maté (2min) Toasted Maté (4min)

150

2000

1000

a

a

b a b

b c

a

c a

c d

d

d

mg/100g

mg/100g

a

a

b

b c

0

50

d

d

d

3,4-diCQA

3,5-diCQA

4,5-diCQA

b

b

a

b b c c

c

b c

a

100 c

c

b

c

c

a

a

d

d

d

0

3-CQA

4-CQA

5-CQA


3-FQA

4-FQA

5-FQA

3-CQL

4-CQL

Distribution of Major Chlorogenic Acids and Related Compounds in

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