Food Research International 60 (2014) 241–245

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Food Research International

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The effect of the duration of infusion, temperature, and water volume on

the rutin content in the preparation of mate tea beverages: An
optimization study
Tayse Ferreira Ferreira da Silveira, Adriana Dillenburg Meinhart,
Cristiano Augusto Ballus, Helena Teixeira Godoy ⁎
Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), 13083-862, Campinas, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The consumption of tea beverages has increased 30% over the last decade, mainly due to the presence of bioactive
Received 17 April 2013 compounds. Mate tea, produced by infusing the leaves and stems of Ilex paraguariensis, is the most widely con-
Accepted 18 September 2013 sumed beverage in Brazil. The present study employed a central composite experimental design to optimize
Available online 26 September 2013
the transfer of rutin from the leaves and stems to the beverage during the infusion process. The optimum condi-
tion was applied to three batches of mate tea beverages from five commercial samples. Analysis of the rutin con-
Keywords:
Rutin
tent was performed by high performance liquid chromatography coupled to a photo diodo array detector. The
Mate tea maximum rutin content in the beverage was obtained when the infusion was performed using 2 g of mate tea
Multivariate optimization added to 100 mL of water at 72 °C and infused for 9 min. The commercial tea beverages prepared under these
HPLC conditions contained from 0.16 to 1.1 mg of rutin in the ready-to-drink product.
© 2013 Published by Elsevier Ltd.

1. Introduction concentrations ranging from 0.3 mg g−1 to 0.5 mg g−1 (Filip, Lopéz,
Giberti, Coussio, & Ferraro, 2001; Ribani, 2006).
Tea-based beverages are among the most popular in the world. Their In vitro and in vivo studies have shown that rutin has potent
consumption increased by approximately 30% over the last decade; this antioxidant, anti-inflammatory, and hepatoprotective activities (Ajay,
increase can be primarily attributed to the in vitro and in vivo studies Anwar-Ul, & Mustafa, 2003; Amira, Rotondo, & Mulè, 2008; Kazłowska,
that demonstrated the inverse relationship between consumption and Hsu, Hou, Yang, & Tsai, 2010; Martins et al., 2009; Moon & Kim, 2012;
the risk of degenerative diseases (Bhattacharya, Mukhopadhyay, & Shenbagam & Nalini, 2011; Yang, Guo, & Yuan, 2008; Zhou, Yao, Cao,
Giri, 2011; Mackay & Blumberg, 2002; Martins et al., 2009). The health Jiang, & Xia, 2006). Additionally, several human studies have highlighted
benefits associated with the intake of these beverages have been corre- the effectiveness of this compound and its derivatives in the treatment
lated with the presence of flavonoids and other phenolic compounds, of vascular diseases (Petruzzellis et al., 2002; Unkauf, Rehn, Klinger, De
which are the most important compounds in tea and have potent anti- La Motte, & Grossmann, 1996). Thus, rutin has been widely used as a
oxidant and free radical scavenging activities (Astill, Birch, Dacombe, component in many drugs intended primarily for patients with vascular
Humphrey, & Martin, 2001; Atoui, Mansouri, Boskou, & Kefalas, 2005; disease (Erlund, 2004; Ihme et al., 1996; Jeong et al., 2009).
Matsubara & Rodriguez-Amaya, 2006). Several univariate studies have shown that the method of preparing
The preparation of mate tea beverages consists of infusing previous- beverages from other teas, like green and white tea, as well as the plant
ly ground and roasted leaves and stems of the Ilex paraguariensis St. Hill growth and processing conditions, directly alters the chemical composi-
plant, which is native to South America (Souza, 2009). In Brazil, tion of teas (Komes, Horzic, Belscak, Kovacevic, & Vulic, 2010;
according to the Brazilian Institute of Geography and Statistics (Instituto Nishiyama et al., 2010; Rusak, Komes, Likíc, Horzic, & Kovac, 2008).
Brasileiro de Geografia e Estatística – IBGE), the per capita consumption The transfer rate of compounds from the tea to the beverage may be af-
of tea in 2008–2009 was 0.500 kg/year, of which 0.482 kg was mate tea fected by the amount of leaves or stems used, the particle size, the water
(IBGE, 2012). The leaves and stems of Ilex paraguariensis are rich in volume, the temperature, the presence or absence of stirring, the dura-
rutin, a flavonoid (flavonol) consisting of a quercetin molecule, linked tion of infusion, and the use of additional ingredients, such as sugar or
to a disaccharide, namely, rhamnose and glucose (Pedriali, 2005). This milk. The effect of different preparation methods on the chemical com-
compound is the primary flavonoid in this plant and is found in position of the beverage ingested by the consumer is important because
the beneficial properties associated with the consumption of these bev-
⁎ Corresponding author. Tel.:+55 19 3521 4024; fax: +55 19 3521 2153. erages can be directly correlated to the components extracted from the
E-mail address: helena@fea.unicamp.br (H.T. Godoy). tea leaves (Astill et al., 2001; Nishiyama et al., 2010).

0963-9969/$ – see front matter © 2013 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.foodres.2013.09.024
242 T.F.F. da Silveira et al. / Food Research International 60 (2014) 241–245

Given that mate tea has high levels of rutin, the consumption of bev- obtained from a mixture of either loose or sachet commercial teas
erages prepared from this plant could be an important source of this from the two most largely commercialized mate tea brands (one unit
compound in the human diet. No studies found in the literature of loose and sachet tea for every commercial brand). The teas had parti-
discussed optimizing the preparation conditions of beverages produced cle sizes of approximately 500 mesh and were studied at that size to
from the hot infusion of mate tea to achieve the maximum efficiency of represent the preparation conditions that would be used by the con-
rutin extraction in the ready-to-drink product. According to Bruns, sumer. To prepare the beverages, 2 g of tea was weighed, which is the
Guadagnini, Scarminio, and Barros Neto (1997) and Ballus, Meinhart, approximate amount contained in a sachet. Hot water was added to
Bruns, and Godoy (2011) multivariate experimental designs can be effi- the tea, with the leaves and stems staying totally immersed in hot
ciently used to optimize processes and to investigate the effects of inde- water, and the infusion remained at rest. After the infusion period, the
pendent variables and the interaction effects among them. teas were immediately cooled to room temperature and transferred to
Thus, the present work aimed to employ a multivariate experimen- volumetric flasks. The contents of the flasks were filtered through filter
tal design to optimize the preparation conditions for mate tea beverages paper (Whatman n° 1), followed by filtration through Millipore 0.45-
and achieve the maximum transfer of rutin from the tea to the beverage μm cellulose filters (Brazil). The filtered solution was injected into the
that is typically prepared for and ingested by the consumer. high performance liquid chromatography apparatus to measure the
rutin content.
2. Materials and methods
2.3. Analysis of the rutin content by high performance liquid chromatography
2.1. Reagents and samples and validation of the method

The rutin standard was purchased from Sigma Chemical (USA). A A high performance liquid chromatography (HPLC) apparatus
stock solution was prepared in methanol at a concentration of (HP 1100, Hewlett Packard, Germany) was used. The HPLC appara-
1 mg mL−1 and was stored at −18 °C. Analytical grade formic acid tus consisted of a diode array detector, quaternary pump system,
was purchased from Ecibra (Brazil), and chromatographic grade meth- autosampler, and column temperature control oven (30 °C). The
anol was purchased from JT Baker (Germany). The mate tea samples chromatographic separation was performed on a Microsorb-MV
were purchased in supermarkets in the city of Campinas, SP, Brazil. C18 reversed-phase column (5 μm × 250 mm × 4.6 mm, VARIAN,
USA). The elution mode was isocratic with a mobile phase consisting
2.2. Multivariate optimization of water - aqueous formic acid (0.1% v/v), and methanol (47.
5:2.5:50.0 v/v/v). The flow rate was maintained at 1 mL min− 1
The present study used a 23 central composite design with 17 trials, with a run time of 10 min. The injection volume was 50 μL. The
including three true replicates at the center point. The effects of three rutin in the sample was identified by comparison with the retention
variables were investigated: the duration of infusion of the leaves, the time of the standard, the UV-visible absorption spectra of the stan-
water temperature, and the water volume used for the preparation of dard, and co-chromatography. The mobile phases were filtered
the beverages, which were highlighted as important determinants of through 0.45-μm cellulose membranes (Millipore, Brazil). The inte-
the component concentration for tea beverages (Astill et al., 2001; grations were performed using Chemstation software (Hewlett
Komes et al., 2010; Nishiyama et al., 2010). The ranges studied were Packard, Germany). Quantitation was performed with an external
from 3.8 to 15.0 min for the duration of infusion (corresponding to standard calibration detected at 360 nm.
levels −1.68 and +1.68), 58.1 to 90 °C for the water temperature, and The chromatographic method was validated using the validation
100 to 400 mL for water volume. These ranges were selected based on rules set by the Brazilian National Health Surveillance Agency (Agência
the values usually employed by consumers when preparing the mate Nacional de Vigilância Sanitária - ANVISA, 2012) with respect to the
tea beverage. The detailed levels are shown in Table 1. The evaluated re- intra-day precision parameters (10 measurements of the same sample
sponse was the rutin content in the final, ready-to-drink beverage. All throughout the day), between-day precision (the quantification of the
experiments were conducted in random order. same sample on three different days), linearity (between 5 equidistant
The mate tea (toasted leaves and stems of I. paraguariensis) used to points at the concentrations of 0.5 and 5.0 mg mL−1, observing the
prepare the beverages used in the central composite design was model-fitting through Analysis of Variance - ANOVA, 95%), and

Table 1
Variables, experimental levels, and concentration of rutin in beverages prepared in the central composite design trials.

Experiment Codified variables Decodified variables Rutin content

(mg of rutin in
Time (x1) (minutes) Temperature (x2) (°C) Volume (x3) (mL) x1 x2 x3
the final beverage)

1 −1 −1 −1 3.8 58.1 160.7 0.192

2 1 −1 −1 12.2 58.1 160.7 0.207
3 −1 1 −1 3.8 81.9 160.7 0.167
4 1 1 −1 12.2 81.9 160.7 0.165
5 −1 −1 1 3.8 58.1 339.3 0,231
6 1 −1 1 12.2 58.1 339.3 0.235
7 −1 1 1 3.8 81.9 339.3 ND b
8 1 1 1 12.2 81.9 339.3 ND
9 0 0 0 8 70 250 0.222
10 0 0 0 8 70 250 0.261
11 0 0 0 8 70 250 0.239
12 −1.68 0 0 1 70 250 ND
13 0 1.68 0 8 50 250 0.149
14 0 0 −1.68 8 70 100 0.189
15 1.68 0 0 15 70 250 0.147
16 0 1.68 0 8 90 250 ND
17 0 0 1.68 8 70 400 0.217
b
ND: content below the detection limit.
 T.F.F. da Silveira et al. / Food Research International 60 (2014) 241–245 243

detection and quantification limits (estimated at 3 and 10 times the Table 3

signal-to-noise ratio, respectively). Analysis of variance of the values for content of rutin of mate tea beverage prepared in the
experimental conditions.

2.4. Application to the samples Factor Sum of square DF Mean square F value p valuea

Regression 0.0853 9 0.0095 5.2778 0.0241

The optimum condition found by the multivariate design was used Residual 0.0124 7 0.0018
to prepare the beverages from 2 g of tea. The rutin content was investi- Lack of fit 0.0117 5 0.0023 6.3333 0.1413
Pure error 0.0007 2 0.0004
gated in beverages obtained from three brands of mate tea samples sold
Total SS 0.0977 16
in individual sachets; three batches of each brand were studied. Similar-
a
ly, three batches of beverages obtained from two brands of mate tea sold significant at the 95% confidence level.

in bulk (loose leaves and stems) were evaluated for a total of 15

samples. lack of fit for the data because the obtained F value (6.34) was lower
than the F5,2 value (19.3). Thus, the whole mathematical model was
2.5. Statistical treatment used to explain the effects of the variables on the system and to predict
the optimum condition for preparing tea beverages to obtain the maxi-
The data analysis was performed using the Statistica 7.0 software mum rutin content.
(Statsoft, USA), as well as the calculus of the critical points, which was The negative quadratic effect of the infusion duration indicated that
important to provide the optimal conditions of preparing the mate tea increasing this variable favored rutin extraction up to maximum level,
beverages. The multivariate optimization results were analyzed using after which there was a decrease. This effect can be observed in the ex-
analysis of variance (ANOVA) to assess the fit of the mathematical periments at the central point and the axial points for this variable: the
model to the experimental data. After the rutin was quantified in the upper axial point, with an infusion duration of 15 min, had half of the
commercial tea samples, the means were compared by ANOVA and rutin concentration obtained at the central point (8 min infusion).
Fisher's test to determine whether there were significant differences Thus, the results showed that prolonged infusion reduced the concen-
among the brands, batches, and types of packaging (sachet or loose). tration of rutin in the beverage. Komes et al. (2010) and Perva-
The samples were considered significantly different at p b 0.05. Uzunalic et al. (2006) used univariate models to study the effect of the
infusion time on the catechin concentration when preparing green
3. Results and discussion tea. Both studies found that longer green tea infusion times favored
the transfer of these compounds to the beverage. However, in agree-
3.1. Validation of the chromatographic method ment with the data obtained in the present study, the authors conclud-
ed that long infusions (over 20 min) resulted in the degradation of
The method for the separation and quantification of rutin was vali- these compounds.
dated and was found to be suitable for quantification according to the The infusion water temperature was another important variable for
ANVISA standards (ANVISA, 2012). For the intra-day precision studies, the efficient transfer of the rutin from the tea to the beverage. The rutin
a relative standard deviation of 2.7% (n = 10) was obtained, whereas concentration continued to increase when using temperatures at the
for the between-day precision, a value of 3.0% (n = 3) was obtained. lower levels, −1 and 0 (58.1 °C and 70 °C). According to Cacace and
The rutin retention times varied by less than 1.0% (n = 10). The method Mazza (2003), raising the temperature increases the solubility and the
was shown to be linear in the 0.5 to 5.0 mg L−1 range (r2 = 0.9996). diffusion coefficient of phenolic compounds, thereby providing higher
The analysis of variance revealed that the regression was significant in extraction rates.
this range and that the mathematical model showed no lack of fit
(p N 0.05), demonstrating that this method is suitable for these mea-
surements. The detection and quantification limits were 0.092 mg L−1
and 0.3 mg L−1, respectively.

3.2. Optimization of the preparation conditions for mate tea beverages

Table 1 shows the studied variables, the experimental levels, and the
concentrations of rutin present in the beverages prepared under the ex-
perimental conditions of the central composite design. The rutin con-
centrations ranged from non-measurable (below the quantification
limit of the method) to 0.26 mg of rutin in the ready-to-drink beverage.
The generated regression coefficients of the model of the rutin con-
centration response are shown in Table 2. The regression for the quadrat-
ic model was significant at the 95% confidence level, and the residuals
were shown to be random with no evidence of heteroscedasticity. The
ANOVA (Table 3) results indicated that the quadratic model showed no

Table 2
Significant regression coefficients for the coded variables of the quadratic model of the
rutin concentration response.

Parameters Coefficient Standard error pa

b0 Mean 0.238 0.011 0.002

b2 Temperature −0.045 0.005 0.013
b21 Time −0.043 0.005 0.017
b22 Temperature −0.043 0.005 0.017
b2 x b3 Temperature x Volume −0.039 0.007 0.029
Fig. 1. Response surface for the rutin concentration (mg of rutin in the final beverage).
a Variables: Time (minutes) × Temperature (°C), with the volume fixed at 100 mL.
significant at the 95% confidence level.
244 T.F.F. da Silveira et al. / Food Research International 60 (2014) 241–245

Table 4 rutin in the beverage. The experimental confirmation was performed

Concentration of rutin in the commercial samples of mate tea beverages. in triplicate, and 0.25 (±0.004) mg of rutin was obtained in the bever-
Rutin content (mg 100 mL−1) age. The hypothesis test used to compare the values showed that at a
95% confidence level, there was no significant difference between the
Brand Batch Package
value predicted by the model and the value obtained experimentally.
Sachet Loose leaves

A 1 0.130 ± 0.002a 0.119 ± 0.006 b 3.3. Application to commercial samples of mate tea
2 0.123 ± 0.003 b 0.101 ± 0.011 b
3 0.118 ± 0.004 c 0.135 ± 0.003 a
Mean 0.124 ± 0,006a, β 0.11 ± 0.013a, υ
The optimum conditions for preparing mate tea beverages to obtain
B 1 0.042 ± 0.004 b 0.047 ± 0.002 b the maximum efficiency of the rutin extraction were applied to com-
2 0.051 ± 0.008 a 0.046 ± 0.021 b mercial samples, and the results are presented in Tables 4. Fig. 2
3 BNF y 0.104 ± 0.002 a shows a chromatography profile of a mate tea beverage prepared
Mean 0.045 ± 0,006b,γ 0.046 ± 0.0007 b, σ
under the optimized conditions.
C 1 0.245 ± 0.008 c UMx
2 0.593 ± 0.019 a The concentration of rutin in 100 mL of the mate tea beverage
3 0.540 ± 0.041 b,C ranged from 0.046 to 0.593 mg for the different commercial samples.
Mean 0.419 ± 0.246α The results showed significant differences (p b 0.05) in the concentra-
Different lower letters, in the columns, for batches of the same brand, indicate significant tion of rutin among beverages prepared from different batches of the
difference (p b 0.05) between samples. same brand. Likewise, the rutin content varied greatly among the bever-
Different lower letters in italic, in the lines for mean of three batches, indicate significant ages obtained from the different brands. The significant differences ob-
difference (p b 0.05) between different packages of commercialization.
served may be attributed to variations in the composition of the plants
Different Greek e letters, in the columns, for mean of three batches, indicate significant
difference (p b 0.05) between samples. due to different growing and processing conditions (Astill et al., 2001;
x
unmarketed by the manufacturer. Heck & Mejia, 2007; Lin, Tsai, Tsay, & Lin, 2003; Mazzafera, 1997;
y
BNF: Batch not found. Pagliosa et al., 2010). Nevertheless, there was no significant difference
among the beverages with respect to the type of packaging (sachet or
loose).
However, when the highest levels of the infusion temperatures, 1.0 Compared to other beverages, the level of rutin in 100 mL of mate tea
and 1.68 (81.9 °C and 90 °C), were used, the concentration of rutin de- beverages found in the present study is much higher than the contents
clined and even reached unquantifiable levels. This negative quadratic present in several beverage source of phenolic compounds as Brazilian
effect is possibly associated with the degradation of this flavonoid. Syrah wine (0.24 mg 100 mL−1, Ballus, Meinhart, Oliveira, & Godoy,
This interpretation is in agreement with the studies of Zhou, Sun, Du, 2012), noni juice (0.188 mg 100 mL−1, Deng, West, & Jensen, 2010),
Liang, and Yang (2000), who evaluated the stability of rutin and found and acerola juice (0.058 mg 100 mL−1, Mezadri, Villaño, Fernandez-
that it begins to degrade at 75 °C. Páchon, García-Parrilla, & Troncoso, 2008). These data show mate tea
Regarding the effect of the interaction between the temperature and beverage as good source of rutin for human diet. In 100 mL of chimarrao,
water volume, it was found that at mild temperatures (levels −1 and the main beverage prepared from yerba mate (which consists of dried
0), increasing the volume of water used to prepare the infusion resulted and ground leaves of Ilex paraguariensis), there is approximately 5 mg
in a beverage with a higher rutin content. However, when both the tem- of rutin (Ribani, 2006). The different processing methods and chemical
perature and water volume were high, lower rutin concentrations were reactions resulting from roasting the leaves of Ilex paraguariensis could
found in the beverage. This degradation effect can be explained by the explain the observed differences (Bastos, Fornari, Queiroz, & Torres,
slower rate of heat loss in beverages prepared with larger volumes of 2006; Matsubara & Rodriguez-Amaya, 2006; Turkumen Erol, Sari,
water (Nishiyama et al., 2010). Çalikoðlu, & Velýoðlu, 2009).
After obtaining a mathematical model with a good fit, it was possible
to optimize the conditions for preparing the beverage by the infusion of 4. Conclusions
mate tea. Fig. 1 shows the response surface generated for the variables
temperature and duration of infusion. The optimum conditions for pre- The use of response surface methodology with a central composite
paring the beverages were as follows: 9.0 min duration of infusion, design was an important tool for evaluating the influence of the studied
water temperature at 72 °C, and water volume of 100 mL. Under variables on the concentration of rutin in mate tea beverages and eluci-
these conditions, the model predicted a yield of 0.21 (±0.027) mg of dating the effects of the interaction among the variables. Using only 17

Fig. 2. Chromatography profile of mate tea water extract. (1) Rutin. Chromatographic conditions: C18 (5 μm × 250 mm × 4.6 mm, VARIAN, USA). Mobile phase: water – aqueous formic
acid (0.1% v/v), and methanol (47.5:2.5:50.0 v/v/v). In isocratic elution and flow rate = 1 mL min−1.
 T.F.F. da Silveira et al. / Food Research International 60 (2014) 241–245 245

experiments, it was possible to determine that the optimal conditions in vivo and protects H9c2 cells against hydrogen peroxide-mediated injury via
for preparing beverages from 2 g of mate tea were as follows: an infu- ERK1/2 and PI3K/Akt signals in vitro. Food Chemistry and Toxicology, 47, 1569–1576.
Kazłowska, K., Hsu, T., Hou, C., Yang, W., & Tsai, G. (2010). Anti-inflammatory properties
sion duration of 9.0 min, water temperature at 72 °C, and water volume of phenolic compounds and crude extract from Porphyra dentate. Journal of
of 100 mL. The beverages prepared under these conditions contained Ethnopharmacology, 128, 123–130.
between 0.16 and 1.1 mg of rutin in 100 mL. Komes, D., Horzic, D., Belscak, A., Kovacevic, K., & Vulic, I. (2010). Green tea preparation
and its influence on the content of bioactive compounds. Food Research
Among flavonoid-rich foods, beverages prepared from the leaves International, 43, 167–176.
and stems of mate tea have significant levels of rutin, and consuming Lin, Y. S., Tsai, Y. J., Tsay, J. S., & Lin, J. K. (2003). Factors affecting the levels of tea polyphe-
these beverages may help supply this substance in the human diet. nols and caffeine in tea leaves. Journal of Agricultural and Food Chemistry, 51,
1864–1873.
Mackay, D. L., & Blumberg, J. B. (2002). The role of tea in human health: An update. 2002.
Acknowledgments Journal of the American College of Nutrition, 21, 1–13.
Martins, F., Martins, F., Suzan, A. J., Cerutti, S. M., Arçari, D. P., Ribeiro, M. L., et al. (2009).
Consumption of mate tea (Ilex paraguariensis) decreases the oxidation of unsaturated
We would like to thank CAPES — Coordenação de Aperfeiçoamento
fatty acids in mouse liver. British Journal of Nutrition, 101, 527–532.
de Pessoal de Nível Superior, and CNPq — Coordenação Nacional de Matsubara, & Rodriguez-Amaya, D. B. (2006). Conteúdo de miricetina, quercetina e
Desenvolvimento Científico e Tecnológico, for their financial support kaempferol em chás comercializados no Brasil. Ciência e Tecnologia de. Alimentos,
to this study. Campinas, 26. (pp. 380–385).
Mazzafera, P. (1997). Mate drinking: Caffeine and phenolic acid intake. Food Chemistry,
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