Daily Intake of Chlorogenic Acids from Consumption of MatÃ© (Ilex

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Daily intakes of chlorogenic acids in Maté (Ilex paraguariensis A.St.-Hil.) traditional beverages consumption. Karimi Sater Gebara, Arquimedes Gasparotto, Patricia Gonçalves Santiago, Claudia Andrea Lima Cardoso, Lauro Mera De Souza, Christine Morand, Telma Aparecida Costa, and Euclides Lara Cardozo Junior J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04093 • Publication Date (Web): 22 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Journal of Agricultural and Food Chemistry

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Daily intakes of chlorogenic acids in Maté (Ilex paraguariensis A.St.-Hil.) traditional beverages consumption.

Karimi Sater Gebaraa; Arquimedes Gasparotto Juniora; Patricia Gonçalves Santiagoa; Claudia Andrea Lima Cardosob; Lauro Mera de Souzac; Christine Morandd; Telma Aparecida Costae; Euclides Lara Cardozo Juniore*;

a

Laboratory of Electrophysiology and Cardiovascular Pharmacology, Health Sciences

College, Federal University of Grande Dourados, Rodovia Dourados-Itahum, Km 12, s/n – J. Aeroporto, 79.804-970 Dourados, MS, Brazil. b

Center for Research on Natural Resources, State University of Mato Grosso do Sul,

Rodovia Dourados-Itahum, Km 12, s/n – J. Aeroporto, 79.804-970 Dourados, MS, Brazil. c

Instituto de Pesquisa Pelé Pequeno Príncipe, Faculdades Pequeno Príncipe, 80.250-060

Curitiba, PR, Brazil. d

INRA, Human Nutrition Unit, UCA, F-63003 Clermont-Ferrand, France.

e

Institute of Biological, Medical and Health Sciences, Universidade Paranaense, Av.

Parigot de Souza, 3636 J. Prada, 85.903-170 Toledo, PR, Brazil.

* Corresponding author: Tel/Fax.: +55 (45) 3277.8500. e.mail: [email protected]

ACS Paragon Plus Environment


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Abstract

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The main objective of the study was to investigate the daily intake of chlorogenic acids

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(CGAs) and methylxanthines by consumers of maté traditional beverages (MTB), terere

4

and chimarrão (Ilex paraguariensis A.St.-Hill). In the studied population (450 citizens

5

from Toledo/PR - Brazil) 63% consume the chimarrão and 37% terere, with weighted

6

mean daily intakes estimated at 648 – 2,160 mL and 244 - 746 mL, respectively. For 100

7

mL of beverage consumed, the average amount of total phenol compounds extracted was

8

673.6 mg for chimarrão and 1,184.9 mg for terere. Regarding CGAs contribution,

9

caffeoylquinic acids (CQAs) constitute about 38.4% for chimarrão and 55.3% for terere,

10

and di-caffeoylquinic acids (diCQAs) represents 61.6 and 44.7% of the extracted

11

compounds, respectively. The daily intake of phenolic compounds by MTB consumers was

12

estimated for chimarrão (512.5 to 1,708.5 mg/day) and terere (583.0 to 1,779.7 mg/day).

13

These results allow us to conclude that MTB are important dietary sources of CGAs,

14

mainly CQAs and di-CQAs.

15 16

Keywords: Ilex paraguariensis; maté traditional beverages; caffeoylquinic acids; maté

17

polyphenols intake.


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Introduction Diets based on polyphenol rich products have been associated with the reduction of 1

20

some chronic diseases in humans, especially cardiovascular diseases (CVD)

.

21

Epidemiological studies have shown that the risk of heart disease can be alleviated with a

22

high consumption of polyphenol rich foods 2. Most cardiovascular diseases (CVD) are

23

caused by complications of atherosclerosis, which is a slow, progressive and inflammatory

24

disease characterized by the development of atherosclerotic plaques in the vascular wall.

25

When associated with arterial hypertension and obesity, dyslipidemias make the

26

development of CVD even more significant 3. These cardiovascular risk factors create a

27

pro-oxidant and pro-inflammatory environment favorable for early development of these

28

diseases.

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Maté (Ilex paraguariensis A. St-Hil.) has been considered an important source of

30

dietary polyphenols with possible interest in the prevention of CVD, as suggested by

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results from in vitro, animal and human studies presented in a recent manuscript review 4.

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In vitro and in vivo research has demonstrated that aqueous extracts from Ilex

33

paraguariensis express high antioxidant activity 5. Moreover, in animal experiments, the

34

consumption of maté has been shown to exert the hypocholesterolemic effect 6, and to

35

attenuate the atherosclerosis development 7, with possible benefits for the cardiovascular

36

system

37

treatment 10, 11.

8, 9

. Some human studies have suggested a potential herbal product in the obesity

38

Recently, mechanisms are proving important properties of maté, which involve

39

cardiovascular health as the antiobesity effect in obese subjects 12, insulin signaling in rats

40

and in human erythrocytes

41

activity in murine model

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biological effects on consumers should not be ruled out, due to the large number of

13-15

, lymphocyte activation in vitro 16, and anti-inflammatory

17

. Therefore, the importance of these compounds and their



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evidence related to the effect of polyphenolic compounds in the prevention of

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cardiovascular diseases.

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Ilex paraguariensis is rich in phytochemicals, especially triterpenic saponins, 18

46

methylxanthines and polyphenols

. The predominant maté phenolic compounds are

47

hydroxycinnamoyl quinic acid esters, classically referred to as chlorogenic acids (CGAs),

48

notably the caffeoylquinic acids (CQAs) and di-caffeoylquinic acids (diCQAs), and

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flavonol glycosides, especially rutin 19. Chlorogenic acids are a family compounds, which

50

are included the caffeoylquinic, p-coumaroylquinic, feruloylquinic, dicaffeoylquinic,

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caffeoylferuloylquinic acids, among other, 20, 21.

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The most common individual caffeoylquinic acids are 3-O-caffeoylquinic acid (3-

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CQA), 5-O-caffeoylquinic acid (5-CQA) and 4-O-caffeoylquinic acid (4-CQA), as well as

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the diCQAs: 3,4-di-O-caffeoylquinic acid (3,4-diCQA), 3,5-di-O-caffeoylquinic acid (3,5-

55

diCQA) and 4,5-di-O-caffeoylquinic acid (4,5-diCQA), and different studies have shown

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that mono and dicaffeoylquinics are the main compounds of the chlorogenic acids family

57

found in maté traditional beverages (MTB)

58

maté has been related to its hight content in phenolic compounds 23. However, few studies

59

have evaluated the absorption, distribution and metabolism of CGAs from MTB, unlike

60

other products such as coffee 24.

22, 23

. The cardiovascular protective activity of

61

Maté is used in South America countries as a tonic and stimulant beverage since the

62

pre-Columbian period. Currently, maté leaves are consumed in a peculiar way by modern

63

and urban populations, despite the originally rural habit 25. The main maté products, such

64

as chimarrão, terere and maté tea, are obtained from the dried and crushed leaves, by

65

different processes that follow the specific regulations of each country. The estimate of

66

chimarrão consumption is of more than 1 liter per day in the Brazilian southern states and

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Uruguay 7. Chimarrão is a hot water beverage and terere is a cold water beverage, both are


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the most common traditional forms of maté consumption 26, and it is relevant to know the

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details of their preparation for understanding the differences in the concentrations of the

70

extracted phytochemical compounds and the contribution in the supply of phenolic

71

compounds. Furthermore, due to the diversity of traditional habits, it remains difficult to

72

determine the average intake of phytochemical compounds from the consumption of MTB.

73

In this study, we aimed to first evaluate the quantity and frequency of maté

74

consumption in the chimarrão and terere forms, and also to assess the impact of socio-

75

economic factors on maté intake. We also reproduced experimentally the conditions used

76

to prepare chimarrão and terere in order to determine the amount of total phenols, mono

77

and di-CQAs, and caffeine extracted and thereby ingested by the maté consumption.

78

Finally, we estimated the daily intake of phytochemicals and characterized the profile in

79

specific compounds, resulting from MTB consumption.

80 81

Materials and methods

82

MTB consumption profile

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A qualitative and quantitative study was conducted to evaluate the profile of MTB

84

consumers and the average intake of phenolic compounds from MTB. Four hundred and

85

fifty-five MTB consumers from the city of Toledo/PR – Brazil were interviewed regarding

86

socioeconomic profile and consumption habits, considering 95% confidence level (p =

87

0.05) and margin of error of 2% (z = 2). The consumers were invited to answer a

88

questionnaire, previously validated, on the socioeconomic profile (sex, age, monthly

89

income, schooling) and consumption habits (type of traditional beverage, daily

90

consumption frequency, container size, amount of maté, beverage volume, number of

91

extractions, number of participants in the round). From these parameters, the average

92

intake of phenolic compounds by the consumers was estimated and the results obtained



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were expressed in mean (mg/day). The interviews took place between July and August,

94

2015 and the study was approved by the Research Ethics Committee (CAAE:

95

22531313.5.0000.0109).

96 97

Content in polyphenols extracted from MTB Samples

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The samples were obtained from commercial products in the city of Toledo/PR -

99

Brazil; three commercial brands of traditional beverages chimarrão (70% of leaves and

100

30% stems) and terere (40% of leaves and 60% stems) were used for the simulated

101

extractions. The extractions were performed in triplicate.

102

Simulated extraction

103

The samples were extracted, simulating the traditional consumption of chimarrão

104

and terere (30 extractions x 3 batches x 3 replicates = 270), based on the methodology

105

adapted by Meinhart et al. (2010). To prepare chimarrão, 85.0 g of maté were added in a

106

traditional medium-sized container called cuia. The preparation of the traditional beverage

107

was made as indicated on the packaging by the manufacturer. 190 mL of water (75 ± 2 °C)

108

was added and after 30 sec. the liquid was extracted with the aid of a vacuum pump

109

coupled to the tube called bomba, and an aliquot was removed for analysis. After two

110

minutes, another 120 mL of hot water was added and the liquid was extracted again. The

111

cuia was supplemented with hot water for thirty times (extractions), thus simulating the

112

aqueous extractions performed by the consumers. To prepare the terere, 50 g of maté were

113

added in an aluminum cup. In this case, 180 mL of water (11 ± 2 °C) was added until

114

complete infusion of the maté for 30 sec., and an aliquot was removed for analysis. The

115

terere samples were also extracted thirty times (extractions) every two minutes, with

116

addition of 100 mL of cold water at each extraction.


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In addition, an experiment was carried out to evaluate the impact of some factors:

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temperature (5 °C – 100 °C), contact time maté/water (0.5 - 4 min.), granulometry (10 - 60

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mesh), stems/leafs ratio (70/30 - 30/70 w/w), on the extraction rate of phenolic compounds

120

from maté consumed in traditional conditions. The procedure used in the extraction

121

process was similar to the previous item for chimarrão and terere, however, the extractions

122

were performed in becker and followed by simple filtration. Six extractions were

123

performed and aliquots were collected for the determination of total phenols. After the

124

extractions, aliquots obtained in each experimental condition were used for determination

125

of total phenolic compounds.

126

Phytochemical analysis

127

Total phenolic content

128

The phenolic compounds were evaluated according to Meinhart et al. (2010) using

129

Folin reagent. One mL of aliquots was diluted to 50 mL. An external calibration curve with

130

pyrogallic acid (Vetec) at concentrations between 4.0 µg/mL and 100.0 µg/mL was used

131

(r2 = 0.9998 for chimarrão and r2 = 0.9999 for terere).

132 133

Caffeoylquinic acids identification UPLC-MS analysis

134

The sample was analyzed by LC-MS in an Ultra Performance Liquid

135

Chromatography (UPLCTM) equipped with a binary pump, column oven and a sample

136

delivery system (Waters Co.). The separation was carried out with a C-18 column, (HSS-

137

T3 - Waters, 100 mm x 2.1 mm, 1.7 µm). Detection was provided by high resolution mass

138

spectrometry (HR-MS). The HR-MS was performed in an LTQ-Orbitrap XL (Thermo

139

Scientific), with electrospray ionization interface (ESI) at atmospheric pressure ionization

140

(API) in the positive and negative ionization modes.

141

Reagents and procedure



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Sample was filtered in 0.22 µm nylon filters with the injection volume of 5 µL. The

143

mobile phase was composed by (A) ultra-pure water (type 1) and (B) acetonitrile (J.T.

144

Baker), both with 0.1% of formic acid (Tedia) (v/v). The gradient used was 0% (initial) to

145

20% in 5 min, then to 70% (13 min) and 0% (13.5 min), flow rate of 400 µL/min, and

146

column temperature of 60 ºC. For sample desolvation, nitrogen at a flow rate of 40

147

arbitrary units (a.u.) in the sheath gas, and 10 a.u in the auxiliary gas were, along with

148

source temperature of 350 ºC. The energies for negative ionization: spray at 3.5 kV, tube

149

lens at -200 V and capillary at -40 V. The mass resolution was set at 15,000 FWHM (at m/z

150

400) in LC-MS mode. External calibration is regularly performed covering m/z 100 – 2000

151

and the acquisition occurred in total ion current (TIC). In the negative ionization, the

152

compounds were fragmented by high-energy collision dissociation (HCD) using helium

153

and energy at 55 eV.

154 155

Caffeoylquinic acids and caffeine quantification HPLC analysis

156

The analysis of the compounds was performed by a Varian (PRO STAR Mod. 210)

157

high performance liquid chromatography coupled with diode array detector (HPLC-DAD),

158

an interphase module Varian (Star 800), and injector (MODEL 410). The chromatographic

159

separation was obtained using a C-18 reversed-phase column (Phenomenex LUNA, 250.0

160

x 4.6 mm x 5.0 µm) with 20 µL injection.

161

Reagents and procedure

162

The samples were filtered in 0.45 µm nylon filters. The elution system consisted of (A)

163

acidified water purified by the Milli-Q system (Millipore Milford, MA) with 0.3% acetic

164

acid and (B) methanol (J.T. Baker). The solvents were run at a flow rate of 1.0 mL/min

165

using the gradient 15- 20% B for 20 min, 20-40% B for 25 min, 40-85% B at 50 min, and

166

85-15% B for 10 min. The column was maintained at 25 °C. Detection was monitored at


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265 nm for caffeine and 325 nm for the CQAs (3-CQA, 5-CQA and 4-CQA) and di-CQAs

168

(3,4-diCQA, 3,5-diCQA and 4,5-diCQA). All samples were run in duplicate and the peak

169

compared with caffeine (y = 803500x + 920; r2 = 0.9996; range from 10.0 to 100.0

170

µg/mL), and chlorogenic acid (y = 495900x - 1460; r2 = 0.9992; range from 10.0 to 100.0

171

µg/mL) standards (Sigma Chemical Co., USA). The detection limit was determined by

172

injecting (n= 5) solutions of each standard solutions of known concentration (0.01 - 0.10

173

µg/mL) and the decreasing the concentrations of the samples until detection of a peak with

174

a signal/noise ratio of 3. The corresponding concentration was considered the minimal

175

detectable concentration. The quantification limit was determined by performing the same

176

methodology and, thus, the quantification limit was defined as the chromatographic peak

177

having a signal/noise ratio of 10. The LOD and LOQ values were 0.023 µg/mL and 0.076

178

µg/mL for chlorogenic acid and 0.018 µg/mL and 0.059 µg/mL for caffeine.

179

Calibration curve was obtained with the mentioned standards after dilution in the

180

mobile phase and submitted to chromatographic separation. The contents of the caffeoyl

181

derivatives were expressed in chlorogenic acid absorbance (mg/100 mL) for CQAs and

182

corrected for di-CQAs using the peak correlation factor (1.458)

183

expressed as mean of each extraction (three batches x three replicates).

184 185

27

. The data were

Results and Discussion MTB consumption profile

186

Maté traditional beverages (MTB) are obtained from the Ilex paraguariensis leaves,

187

and its use is strongly linked to rural customs of South America countries. The origin of

188

the maté consumption refers to pre-Columbian populations (Guaranis), and the basis of the

189

maté myth of origin invariably refers to a "gift of the gods to assuage the grief and fatigue

190

and the bitter hours of old age". Only few studies have evaluated the consumption habits

191

and the impact on phytochemical intake by the MTB consumers. Previous reports have



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estimated the daily consumption of maté leaves in 19.5 g/person/day in Uruguay and 14

193

g/person/day in Argentina

194

extracted from the MTB were also calculated from the use of traditional consumption

195

simulation

196

compounds ingested by MTB consumers. Therefore, our first objective was to evaluate the

197

profile of phenolic compounds provided by the maté consumption, to quantify the intake of

198

polyphenolic compounds and assess the factors that may influence the level of intake in

199

these consumers.

25

. The amount of methylxanthines and phenolic compounds

26

. However, none of these studies investigated the profile of phenolic

200

A significant sampling (n = 455) of MTB consumers was interviewed (Table 1), and it

201

was observed that chimarrão consumers were predominantly women (62.9%), thirty years

202

or more (73.1%), earn less than a $ 1,069.00 (87.5%) and middle level of schooling

203

(55.2%). This profile was quite similar for terere consumers. Indeed, these latter were

204

predominantly women (68.6%), between fifteen to thirty years (81.1%), earn less than a $

205

1,069.00 (90.0%), and middle level of schooling (78.7%). Although consumption

206

originates in the rural environment, the urbanization of these regions has led to adaptations

207

to traditional consumption. We have shown that chimarrão (hot water) is a popular

208

consumer product for people over 30 years old, while terere (cold water) is a product

209

consumed by younger people (15 to 30 years old) with a difference in levels of schooling

210

(55.2 and 78.7%, respectively). This pattern of consumption is representative of the

211

western region of Paraná state (southern Brazil) and may also be indicative of the

212

consumption patterns in other countries. It was possible to observe that in both cases the

213

consumption of chimarrão and terere is a collective habit, wherein chimarrão is consumed

214

in average by 3.1±1.2 people and terere by 3.6±1.5 people/group (Table 1).

215

Estimation of MTB daily intake


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In this work, we determined the average intake of each beverage consumed from

217

results of the survey carried out on consumers. The diversity of consumption habits

218

demonstrated in ours and other studies 26 may involve variations in the recipient size (cuia,

219

guampa), number of rounds, and social habits related to the consumption. Table 2 shows

220

the traditional consumption habits used to evaluate the amount of MTB consumed. It was

221

possible to observe that most of the interviewees consume chimarrão in a medium-sized

222

container (85 g / 120 mL), daily, one to two times a day, and where they execute about 7.8

223

± 4.2 successive extractions. The terere is consumed preferably in a medium-sized

224

containers (50 g / 100 mL), one to two times a day, two to four times a week, and which

225

perform 7.7 ± 3.9 extractions. To estimate chimarrão consumption was multiplied:

226

Volume (120 mL) x Average times / day (1.5) x [Average times per week / weekdays] (7/7

227

= 1.0) x Minimum or maximum times per rounds (3.6 or 12). In the same way to estimate

228

terere consumption was multiplied: Volume (100 mL) x Average times / day (1.5) x

229

[Average times per week / weekdays] (3/7 = 0.43) x Minimum or maximum times per

230

rounds (3.8 or 11.6). The evaluation of the habits of consumers allows estimating a daily

231

consumption from 648 to 2,160 mL for chimarrão and 244.3 to 745.7 mL for terere.

232

Caffeoylquinic acids identification

233

Maté is an important source of methylxanthines and phenolic compounds; the main

234

compounds extracted in traditional consumption being caffeine and chlorogenic acids 28. In

235

order to identify compounds of the caffeoylquinic family, a sample of the extracts obtained

236

in traditional consumption was analyzed by UPLC-MS. The secondary metabolites were

237

observed, namely those from the series of isomeric mono-caffeoylquinic and di-

238

caffeoylquinic acids. The mono-caffeoylquinic acids compounds were fragmented,

239

producing 3 deprotonated ions at m/z 353.0876 [M-H]- with fragment-ions at m/z 135.044,

240

173.045, 179.034 and 191.056. Mono-caffeoylquinic acids were identified as 3-O-



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caffeoylquinic acid (3-CQA), 5-O-caffeoylquinic acid (5-CQA) and 4-O-caffeoylquinic

242

acid (4-CQA). The series of di-caffeoylquinic acids were observed at m/z 515.119 [M-H]-,

243

confirmed by fragment-ions at m/z 135.044, 173.045, 179.034, 191.056 and 353.087. Di-

244

caffeoylquinic acids were identified as 3,4-di-O-caffeoylquinic acid (3,4-diCQA), 3,5-di-

245

O-caffeoylquinic acid (3,5-diCQA) and 4,5-di-O-caffeoylquinic acid (4,5-diCQA). These

246

fragments appeared at different ratios, being characteristic for each isomer, as described in

247

previous work

248

confirmed by the UV absorption spectra of the samples analyzed in HPLC-DAD

249

(unpublished data) and literature data. The UV spectra showed maximum at 328-330 nm

250

and a shoulder at 266-272 nm, typical of caffeoylquinic acids. CQAs and di-CQAs were

251

reported as major phenolic compounds in different beverages obtained from yerba-maté 23,

252

29-31

253

3,5-di, and 4,5-diCQA) was the same in the caffeoylquinic acids identification (UPLC) and

254

quantification experiments (HPLC). The presence of glycosylated flavonoids as well as

255

other hydroxy cinnamoylquinic acids was reported in other studies

256

quantification of these compounds was not addressed in the present work.

257

18

. The identification of the mono and di-caffeoylquinic isomers was also

. The elution sequence of the mono (3-acyyl, 5-acyl, and 4-acyl) and di-CQA (3,4-di,

23, 28

, but the

Caffeoylquinic acids and caffeine quantification

258

The diversity of the consumption habits of MTB is a factor that difficult the

259

quantification of the phytochemicals consumed. The consumption is carried out with

260

successive extractions from the same raw material (matrix), and different factors can

261

influence this process. Caffeine, mono and di-caffeoylquinic isomers were quantified by

262

HPLC-DAD (Fig. 1). The content of theobromine was evaluated (data not shown) and the

263

concentrations found were very small. The presence of impurities with the same absorption

264

spectrum in some analyzed peaks of the chromatogram, as well as the use of peak

265

correlation factor for quantification of di-caffeoylquinic acids may be reasons to


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overestimate the values obtained, but to our point of view do not compromise the general

267

estimates of the daily intake of the analyzed compounds. The quantities obtained from

268

successive extractions were added (Table 3), where the three first extractions, the sum of

269

eight extractions (∑8) and the sum of thirty extractions (∑30) were observed. The ∑8 was

270

selected because it was the average of rounds observed in the interviews (Table 2).

271

Extraction curves of caffeine (Fig. 2A) and phenolic compounds (Fig. 2B) were

272

performed using a simulation of the traditional technique. The extraction curves obtained

273

presented a pattern similar to that of other studies 26, 32. The extraction curves of chimarrão

274

presented higher phytochemical contents in the first extraction, later it decrease and

275

remains with small variations until the thirtieth extraction. Terere show an increase in the

276

phytochemical content in the second and third extractions, then it declines abruptly

277

between the seventh and eighth extractions, which coincide with the average number of

278

extractions evaluated by the consumption habits (Table 2) and decays gradually until the

279

thirtieth extraction. The amount of the thirty extractions (∑30) performed in the simulation

280

of chimarrão yielded 6.3 mg of caffeine and 673.6 mg of total phenolic compounds and for

281

terere yielded 68.3 mg of caffeine and 1,184.9 mg of total phenolic compounds (Table 3).

282

The main phenolic compounds present in the extractions were also quantified. The mono-

283

caffeoylquinic (3-CQA, 4-CQA and 5-CQA) and di-caffeoylquinic (3,4-diCQA, 3,5-

284

diCQA and 4,5-diCQA) acids identified in the extracts as described above were quantified

285

in HPLC-DAD and expressed as chlorogenic acid and the results presented in Table 3. The

286

∑(mono-CQAs) constitute about 38.4% for chimarrão and 55.3% for terere, and the ∑(di-

287

CQAs) represents about 61.6 and 44.7% of the total phenolic compounds extracted,

288

respectively. The ∑(mono and di-CQAs) represented 87% of total phenolic in terere and

289

only 25% in chimarrão.

290

process, that will be discussed, have a high influence in the extractive capacity. These

We observe that particularities of the chimarrão extractive



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results demonstrated that terere presents a higher extractive capacity than the chimarrão

292

for both caffeine (Fig. 2A) and phenolic compounds (Fig. 2B). A small difference in the

293

profile of phenolic compounds extracted was also observed, with a higher concentration of

294

di-CQAs in chimarrão and a higher concentration of CQAs in tererê.

295

The amount of the first eight extractions (∑8) for chimarrão yielded 1.9 mg of

296

caffeine and 245.2 mg of total phenolic compounds, respectively 30.5 and 36.4% of the

297

total extractions (∑30). The first eight extractions of terere obtained 48.7 mg of caffeine

298

and 859.2 mg of total phenolic compounds, respectively 71.3 and 72.5% of total (Table 3).

299

The profile of phenolic compounds extracted in the first eight extractions (∑8) was similar

300

to the one presented previously, with a higher concentration of di-CQAs in chimarrão, and

301

higher concentration of mono-CQAs in terere.

302 303

Factors influencing the extracted content of phenolic compounds Maté is a rich source of phenolic compounds and different factors can modulate the 33

304

qualitative and quantitative content of the extracted compounds

. Simulations of MTB

305

preparation were performed to evaluate the influence of some factors involved, including

306

temperature, contact time maté/water, granulometry and stems/leafs ratio in the extraction

307

rate of total phenolic compounds. The results show the sum of six extractions (∑6) in a

308

similar way to the quantity defined in the habits of consumption. The conditions of 75 °C

309

(temperature), 30 sec. (time), 60 mesh (granulometry) and 30/70 (stems/leafs ratio) for

310

chimarrão, and 5 ºC (temperature), 30 sec. (time), 30 mesh (granulometry) and 60/40

311

(stems/leafs ratio) for terere were used as the standard extraction. The coefficient of

312

correlation with the content of phenolic compounds was elevated for temperature and

313

granulometry in both beverages (Table 4), while the time was of lowest correlation.

314

The granulometry, within a range of 10 to 60 mesh, was the factor of greatest impact

315

on the extraction capacity of phenolic compounds for both beverages, chimarrão (6.8 to


Page 15 of 34


15 316

44.6 mg / 100 mL) and terere (4.0 to 21.4 mg / 100 mL) when using the procedure of

317

traditional consumption. The temperature was the other factor with the greatest influence

318

on the extraction capacity of total phenolic compounds, chimarrão (32.7 to 54.0 mg/100

319

mL) and terere (8.2 to 22.6 mg/100 mL) for a range of 5 to 100 ºC. The extraction time

320

and the stems/leafs ratio had less influence.

321

Daily intakes of phenolic compounds from MTB

322

Chimarrão and terere are MTB used as stimulants due to the presence of

323

methylxanthines 34, furthermore, maté is a source of CQAs and diCQAs and these are the

324

major constituents of the phenolic fraction. Other groups that comprise the phenolic

325

fraction are dimeric cafeic acids, feruloylquinic acids, p-coumaroylquinic acids and

326

flavonoids

327

and di-caffeoylquinic acids (3,4-di-CQA, 3,5-di-CQA and 4,5-diCQA) 30. The daily intake

328

of phenolic compounds was calculated from the quantification of the compounds in the

329

beverages and applying the data on the MTB consumption. The contents extracted from

330

phenolic compounds were considered by other authors 26, but our objective was to present

331

an estimation of the daily intake of phenolic compounds from MTB and to determine how

332

these intakes can be influence by factors related to the preparation of the beverages. The

333

estimated intake of phenolic compounds for chimarrão consumers was 512.5 to 1,708.5

334

mg/day, 38.8% refers to mono-CQAs and 61.2% to di-CQAs, and terere consumers was

335

583.0 to 1,779.7 mg/day, being that 55.3% refers to mono-CQAs and 44.7% to di-CQAs.

336

These results were obtained from the multiplication of the average volume per day (Table

337

2) by the total phenolic content (∑8) (Table 3), and dividing by the number of people per

338

consumption group, considering the collective habit for each beverage (3.1 people/group

339

for chimarrão and 3.6 for terere).

23, 28

. The main phenolic compounds are mono (3-CQA, 5-CQA and 4-CQA)



Page 16 of 34

16 340

The concentrations of caffeine and phenolic compounds in chimarrão beverage were

341

lower than that terere, and also lower than the results presented in previous studies 26. For

342

the preparation of chimarrão it was observed that the product is not completely moistened

343

with the extraction water. The small particle size of the product forms a barrier that hinders

344

the passage of water and reduces the extraction capacity. Conversely, the terere had larger

345

particle size and these were completely immersed in water, providing greater contact and

346

consequently greater extractive capacity. The lower contents of total phenolic, CQAs and

347

diCQAs to chimarrão (Table 3) may be a result of this effect.

348

Although the terere presents greater capacity of phenolic compounds extraction, the

349

daily intake did not present any significant difference considering consumption habits. The

350

average volume consumed per day of chimarrão is superior to terere, because the

351

frequency of consumption. From this study it was possible to quantify the daily intake of

352

phenolic compounds by consumers of MTB, besides identifying which compounds are

353

predominant. Our results demonstrate that MTB are important sources of caffeoylquinic

354

acids, highlighting a small difference in the profile of mono and di-caffeoylquinic acids

355

between the two beverages.

356

Other stimulant beverages also contain phenolic compounds, for comparison purposes,

357

the amount of the mono-CQAs, di-CQAs and caffeine presents in different coffees, varies

358

from 135.1 to 187.2 mg / 100 mL, 8.3 to 11 mg / 100 mL and 48.2 to 57.7 mg / 100 mL,

359

respectively 35. Considering that the extracted amount (∑30) of the compounds chimarrão

360

can provide 65.6 mg / 100 mL, 105.3 mg / 100 mL and 6.3 mg / 100 mL and terere, 575.4

361

mg / 100 mL, 460.2 mg / 100 mL and 68.3 mg / 100 mL, significant difference is observed

362

in the phytochemical content between the beverages. From the values obtained from the

363

extractions, and considering the consumption habits, it is possible to infer that the MTB

364

can provide a higher content of phenolic compounds, with greater importance of the CQAs


Page 17 of 34


17 365

and specifically higher contribution of diCQAs. Terere beverage presents a higher

366

extraction capacity than chimarrão for both phenolic compounds and methylxanthines. As

367

expected, the first extractions are more concentrated than the final ones. It has also been

368

noted that chimarrão has a higher concentration of di-CQAs and terere has more mono-

369

CQAs.

370

An aspect to be considered is that the experimental approach used here is not

371

conclusive as to the amount of compounds made available to consumers. Intervention

372

studies are indicated to evaluate the bioavailability and bioactivity of the components after

373

drinking beverages. Further research is needed to verify the bioavailability of mono and

374

dicaffeoylquinic compounds from the raw material used in MTB. The bioavailability has

375

recently been evaluated in animals

376

existing knowledge on the efficiency of these compounds from maté consumption in the

377

maintenance of health, especially in the prevention of CVD. However, after our evaluation

378

we have a closer estimate of the daily intake of caffeoylquinic acids available to the MTB

379

consumers.

36

, but human studies are still necessary to strengthen

380

The beneficial biological effects of CGAs have already been reported, and it is

381

therefore relevant to investigate whether the extracted amount of these compounds in MTB

382

contribute to a cardiovascular protective activity in the concentrations normally consumed.

383

Our objective was to evaluate the daily intakes of phenolic compounds, however it is

384

known that only a small amount of these compounds are absorbed 37, 38. It is also necessary

385

to evaluate the participation of the metabolic processes and the gut flora in the

386

transformation of these compounds before reaching the tissues. The presence of

387

considerable amounts of di-CQAs makes the MTB as single sources, unlike other

388

beverages where CQAs predominates.

389



Page 18 of 34

18 390

Abbreviations Used

391

API = Atmospheric Pressure Ionization

392

b.m.w. = brazilian minimum wage

393

CQAs = Caffeoylquinic acids

394

CGAs = Chlorogenic acids

395

CVD = Cardiovascular Diseases

396

ESI = Electrospray Ionization Interface

397

HCD = High-Energy Collision Dissociation

398

HPLC-DAD = High Performance Liquid Chromatography coupled with Diode Array

399

Detector

400

HR-MS = High Resolution - Mass Spectrometry

401

LC-MS = Liquid Chromatography - Mass Spectrometry

402

MTB = Maté Traditional Beverages

403

m/z = mass-to-charge ratio

404

TIC = Total Ion Current

405

UPLC = Ultra Performance Liquid Chromatography

406

UV = Ultraviolet

407

Σ(mono-CQAs) = Sum of mono-caffeoylquinic acids (3-CQA; 5-CQA and 4-CQA)

408

∑(di-CQAs) = Sum of di-caffeoylquinic acids (3,4-diCQA; 3,5-diCQA and 4,5-diCQA)

409

∑(mono and di-CQAs) = Sum of mono and di-caffeoylquinic acids

410

∑6 = Sum of the first six extractions in laboratory simulation

411

∑8 = Sum of the first eight extractions in MTB

412

∑30 = Sum of thirty extractions in MTB

413 414


Page 19 of 34


19 415

Acknowledgements

416

We are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico

417

(CNPq-Brazil), Universidade Paranaense (UNIPAR) and Universidade Federal da Grande

418

Dourados (UFGD). All authors declare no financial/commercial conflicts of interest

419

regarding the information in this article.

420



Page 20 of 34

20 421

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composition and blood chemistry in healthy, overweight subjects--a randomised double-

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L.; Schinella, G. R.; Mosca, S. M., Effect of an Ilex paraguariensis (yerba mate) extract on

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Baratto, B.; Fröde, T. S.; Reginatto, F. H.; Dalmarco, E. M., The anti-inflammatory effect

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of Ilex paraguariensis A. St. Hil (Mate) in a murine model of pleurisy. Int

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Immunopharmacol. 2016, 36, 165-72.

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Gorin, P. A. J.; Sassaki, G. L., UPLC-PDA–MS evaluation of bioactive compounds from

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leaves of Ilex paraguariensis with different growth conditions, treatments and ageing.

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Iacomini, M.; Sassaki, G. L., Comprehensive analysis of mate (Ilex paraguariensis)

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compounds: Development of chemical strategies for matesaponin analysis by mass

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Portela, J. L.; Soares, D.; Rosa, H.; Roos, D. H.; Pinton, S.; Ávila, D. S.; Puntel, R.

Muñoz-Culla, M.; Sáenz-Cuesta, M.; Guereca-Barandiaran, M. J.; Ribeiro, M. L.;

Luz, A. B.; da Silva, C. H.; Nascimento, M. V.; de Campos Facchin, B. M.;

Dartora, N.; de Souza, L. M.; Santana-Filho, A. P.; Iacomini, M.; Valduga, A. T.;

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analysis

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Acids Commonly Known as Chlorogenic Acids. J Agric Food Chem. 2017, 65 (18), 3602-

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Baratto, B.; Fröde, T. S.; Sandjo, L. P.; Dalmarco, E. M.; Reginatto, F. H., Qualitative and

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PDA and QTOF-MS. Data Brief. 2016, 8, 295-9.

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constituents of mate (Ilex paraguariensis, St. Hil.) and its antioxidant activity compared to

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coffee chlorogenic acids in humans. Food Funct. 2014, 5 (8), 1727-37.

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R.; Cerro-Quintana, R. S.; Teixeira-Filho, J.; Godoy, H. T., Methylxanthines and phenolics

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content extracted during the consumption of mate (Ilex paraguariensis St. Hil) beverages. J

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Agric Food Chem. 2010, 58 (4), 2188-93.

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compounds: Development of chemical strategies for matesaponin analysis by mass

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spectrometry. J. Chromatogr. A. 2011, 1218, 7307-7315.

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LC-MSn of the chlorogenic acids and hydroxycinnamoylshikimate esters in mate (Ilex

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Major Chlorogenic Acids and Related Compounds in Brazilian Green and Toasted Ilex

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da Silveira, T. F. F.; Meinhart, A. D.; de Souza, T. C. L.; Teixeira Filho, J.; Godoy,

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Souza, L. M. D.; Dartora, N.; Scoparo, C. T.; Cipriani, T. R.; Gorin, P. A. J.;

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Page 26 of 34

26

Figure captions Figure 1. Representative HPLC-DAD chromatogram of Ilex paraguariensis extracts, signal at 325 nm and 265 nm. Chromatographic conditions are specified in Materials and methods. The indicated compounds are: (1) 3-O-caffeoylquinic acid (3-CQA), (2) 5-Ocaffeoylquinic acid (5-CQA), (3) 4-O-caffeoylquinic acid (4-CQA), (4) 3,4-di-Ocaffeoylquinic acid (3,4-diCQA), (5) 3,5-di-O-caffeoylquinic acid (3,5-diCQA), (6) 4,5-diO-caffeoylquinic acid (4,5-diCQA) and (7) caffeine.

Figure 2. Caffeine (A) and Phenolic content (B) - Total Phenolic (), ∑(Mono and dicaffeoylquinic acids) (), ∑(Di-caffeoylquinic acids) (),∑(Mono-caffeoylquinic acids) (▲) - in the aqueous extracts of chimarrão and terere.


Page 27 of 34


27

Tables Table 1. Socioeconomic Profile of MTB Consumers (n = 455) Toledo – PR, Brazil, 2015. Variables

Chimarrão (n = 286)

Terere (n = 169)

n

%

n

%

Female

180

62.9

116

68.6

Male

106

37.1

53

31.4

15 - 30 years

77

26.9

137

81.1

> 30 years

209

73.1

32

18.9

Primary school

71

24.8

19

11.2

Secundary school

158

55.2

133

78.7

Graduate

57

20.0

17

10.1

1–4

250

87.5

152

90.0

>4

36

12.5

17

10.0

Gender

Age

Schooling

Monthly personal income (b.m.w.) a

People per consumption group 3.1 ± 1.2 a

3.6 ± 1.5

brazilian minimum wage: US$ 267.88 at the time the baseline study was conducted.



Page 28 of 34

28

Table 2. Habits Profile of Maté Traditional Beverages Consumers. (% = Percentage of Consumers Interviewed). Container Traditional beverage

Chimarrão

Terere

Consumption

Amount of maté / water (%)

Times per day (%)

Times a week (%)

< 85 g / 120 mL (92.3)

One / two (80.5)

Everyday (61.9)

> 85 g / 120 mL (7.7)

Three / four (19.5)

Two to four (25.9)

< 50 g / 100 mL (97.7)

One / two (94.7)

Everyday (19.5)

> 50 g / 100 mL (2.3)

Three / four (5.4)

Two to four (80.5)

a

Extractions

Beverage a

Times / round (n)

Average volume per day (mL) 648.0

7.8 ± 4.2

2,160.0 244.3

7.7 ± 3.9

745.7

Estimated from calculation: volume x average daily consumption x average weekly consumption x. minimum / maximum extractions.


Page 29 of 34


29

Table 3. Amount of Caffeine and Phenolic Compounds Extracted of Maté Traditional Beverages (MTB). Extraction (mg/100 mL) Beverage

Chimarrão

Terere

Compounds

1st

2nd

3rd

∑8

∑30

Caffeine

1.2

0.2

0.1

1.9

6.3

∑(mono-CQAs)

12.1

3.2

0.8

20.6

65.6

∑(di-CQAs)

18.8

3.9

0.5

29.5

105.3

∑(mono and di-CQAs)

30.9

7.1

1.3

50.2

170.9

Total Phenolic

82.2

42.5

27.3

245.2

673.6

Caffeine

2.8

9.2

7.4

48.7

68.3

∑(mono-CQAs)

36.9

110.6

63.2

457.7

575.4

∑(di-CQAs)

20.7

60.2

67.3

351.0

460.2

∑(mono and di-CQAs)

57.5

170.8

130.5

808.7

1,029.1

Total Phenolic

92.9

181.4

156.1

859.2

1,184.9



Page 30 of 34

30

Table 4. Correlation Coefficient of Factors that Influence Total Phenolic Content (∑6) of MTB. Temperature

Time

Granulometry

Stems/Leafs Ratio

Chimarrão

0.996

0.037

0.950

0.911

Terere

0.974

-0.161

0.952

0.829


Page 31 of 34


31

Figures Figure 1. 325 nm

265 nm



Page 32 of 34

32

Figure 2.

Caffeine (mg/100 mL)

10.0

(A) Terere

7.5

Chimarrão 5.0

2.5

0.0 0

5

10

15 Extractions


20

25

30

Page 33 of 34


33

Phenolic content (mg /100 ml)

(B)

Terere

200.0

Total Phenolic

150.0

∑(Mono and di-O-caffeoylquinic acids) ∑(Di-O-caffeoylquinic acids) 100.0

∑(Mono-O-caffeoylquinic acids)

50.0

0.0 0

5

10

15

20

25

30

Extractions

Phenolic content (mg /100 ml)

100.0

Chimarrão Total Phenolic

75.0

∑(Mono and di-O-caffeoylquinic acids) ∑(Di-O-caffeoylquinic acids)

50.0

∑(Mono-O-caffeoylquinic acids)

25.0

0.0 0

5

10

15

20

Extractions


25

30


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34

Graphic for table of contents


Daily Intake of Chlorogenic Acids from Consumption of MatÃ© (Ilex

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