Research Article

Received: 24 November 2016 Revised: 2 February 2017 Accepted article published: 11 February 2017 Published online in Wiley Online Library: 9 March 2017

(wileyonlinelibrary.com) DOI 10.1002/jsfa.8255

Guayusa (Ilex guayusa L.) new tea: phenolic

and carotenoid composition and antioxidant
capacity
Almudena García-Ruiz,a* Nieves Baenas,b Ana M Benítez-González,c
Carla M Stinco,c Antonio J Meléndez-Martínez,c Diego A Morenob
and Jenny Rualesa

Abstract
BACKGROUND: Guayusa (Ilex guayusa Loes) is an evergreen tree native of South America that grows particularly in the upper
Amazon region of Ecuador. For its health benefits, it has been cultivated and consumed since ancient times by Amazon
indigenous tribes.

RESULTS: A total of 14 phenolic compounds were identified and quantified. Chlorogenic acid and quercetin-3-O-hexose were the
main representatives of the hydroxycinnamic acids and flavonols, respectively. Five carotenoids were identified, showing lutein
the highest concentration. Guayusa leaves revealed high antioxidant capacity determined by two analytical methods, DPPH
and ORAC. The industrial processing applied to the leaves modified the composition of bioactive compounds and antioxidant
capacity of guayusa. In general, blanched guayusa retained the concentration of phenolic compounds and some carotenoids and
similar antioxidant capacity as untreated green leaves. In contrast, fermentation reduced the content of bioactive compounds
and showed the lowest antioxidant capacity.

CONCLUSION: Therefore, blanched guayusa has potential for product development as a functional ingredient in the food
industry.
© 2017 Society of Chemical Industry

Keywords: Ilex guayusa; phytochemical composition; antioxidant capacity; industrial processing

INTRODUCTION antioxidant activity, which is important in countering oxida-

Guayusa (Ilex guayusa Loes) is an evergreen tree native of South tive stress.7 In addition, carotenoids have also been associated
America that grows between southern Colombia and northern with numerous beneficial effects on human health: enhancement
Peru, particularly in the upper Amazon region of Ecuador. The of the immune response and reduction of the risk of degener-
distribution of this species is from sea level to 2000 m.1 Guayusa is ative diseases such as cancer, cardiovascular diseases, cataract,
and macular degeneration. The action of these compounds
distantly related to the ‘Yerba Mate’ or ‘Mate’ (Ilex paraguariensis),
against diseases has been primarily attributed to their antioxidant
both plants being described as sources of caffeine.2
capacity, but could also be due to other mechanisms, like the
For its health benefits, such as alleviating diverse pains and
absorption of blue light in the retina or actions at the molecu-
preventing undesirable central nervous system effects, guayusa
lar level.8 Thus, it is important to characterize the phenolic and
has been cultivated and consumed since ancient times by Ama-
carotenoid composition in order to correlate their benefits with
zon indigenous tribes, particularly Kichwa and Shuar, forming an human health. In this sense, the characterization of these bioactive
important part of their rituals and ceremonies.2,3 The leaves are
prepared as the tea, that is to say, as a hot infusion of the dried
and minced leaves and twigs of I. guayusa, its flavor being naturally ∗ Correspondence to: A García-Ruiz, Department of Food Science and Biotech-
smooth and never bitter, with a rich and earthy aroma and slightly nology, Escuela Politécnica National, P.O. Box 17–01, 2759 Quito, Ecuador.
sweet finish. Nowadays, the consumption of guayusa is expanding, E-mail: [email protected]
being marketed as an infusion, energy drink and/or ingredient in
a Department of Food Science and Biotechnology, Escuela Politécnica National,
other products in Ecuador, USA, China and Europe.1,4 This reflects Quito, Ecuador
the interest of consumers to incorporate new foods or new food
ingredients with health promoting effects. b Phytochemistry Lab., Department of Food Science and Technology,
CEBAS-CSIC, Espinardo, Murcia, Spain
The major constituents of Ilex spp. are phenolic compounds.5,6
These compounds are of great interest since they are attributed c Food Colour & Quality Lab., Department of Nutrition & Food Science, Facultad
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various beneficial biological effects; the most relevant is de Farmacia, Universidad de Sevilla, Spain

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 www.soci.org A García-Ruiz et al.

compounds requires advanced analytical techniques. Specifically, controlled conditions. The green leaves were fresh leaves just
reverse-phase liquid chromatography (RP-LC) is the most common harvested; this material was stored at −20 ∘ C until freeze-dried.
method for the separation and analysis of phenolic compounds Blanched and fermented guayusa were prepared in the manufac-
while UV detection and mass spectrometry are the techniques turing plant of RUNA Foundation, according to standard protocols
commonly used for identification and quantification of phenolic in this company. Then, leaves of blanched and fermented guayusa
compounds in plants.9 Rapid resolution liquid chromatography were freeze-dried. Finally, leaves of green and processed guayusa
(RRLC) is employed for the characterization of carotenoids.10 Fur- were crushed in a mortar.
thermore, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the oxygen
radical absorption capacity (ORAC) have been frequently used to Extraction procedure
estimate antioxidant capacities in plants and foods.11 For the total phenolics content (TPC) and HPLC analysis, each
On the other hand, industrial processing of I. guayusa involves sample (100 mg) was mixed with 1 mL of methanol/water (70:30,
steps such as blanching or fermentation which may cause some v/v) and was acidified with 1% of formic acid. Then, the samples
changes in the profile and concentration of their bioactive com- were vortexed and sonicated in an ultrasonic bath for 60 min.
pounds (i.e. polyphenols and carotenoids) that could modify its The samples were kept at 4 ∘ C overnight and sonicated again
biological activities. Blanching is a process of preheating the prod- for 60 min. A centrifugation (model EBA 21, Hettich Zentrifugen;
uct by immersion in water or steam whose main objective is the Tuttlingen, Germany) step (9500 × g, 5 min) was used to separate
inactivation of the enzymes present in foods.12 Meanwhile, fer- the supernatant from the solid residue. This supernatant was
mentation in guayusa manufacturing does not involve a microbial filtered through a 0.45 μm PVDF filter (Millex HV13; Millipore,
process, as in wine and beer, but chemical changes in their leaves Bedford, MA, USA) and stored at 4 ∘ C before the analyses were
catalyzed by endogenous enzymes. performed.
The characterization of bioactive compounds like carotenoids For rapid resolution liquid chromatography (RRLC) analysis,
and phenolics in edible native plants and the effect on them of 10 mg of homogenized freeze-dried powder from the samples
processing is always a timely research topic.13,14 were used for the extractions. The powder was gently mixed
The interest in natural antioxidants found in plants has also with 1 mL of the MilliQ-water and then vortexed and centrifuged
increased due to the worldwide increase in using plant extracts as to remove the aqueous phase at 18 000 × g for 3 min. Subse-
additives in food. Taking into account this consideration, the main quently, 1 mL of extracting solvent (hexane/acetone, 1:1 v/v) was
objective of this study was to characterize for the first time the added, the mixture was vortexed and then centrifuged for 3 min at
phenolic and carotenoid composition and antioxidant capacity 18 000 × g. After recovering the colored fraction, a further 500 mL
of I. guayusa. Additionally, and in order to evaluate the effect of of extracting solvent was added, and the mixture was vortexed and
industrial processing on the composition of bioactive compounds finally spun as explained before. The pooled organic colored frac-
of guayusa, the carotenoid and phenolic fraction as well as the tions were eventually evaporated to dryness in a vacuum concen-
antioxidant capacity of blanched and fermented guayusa were trator at a temperature below 30 ∘ C and stored under N2 at −20 ∘ C
analyzed. until analysis.

Total phenolics content

MATERIAL AND METHODS The determination of the TPC using the colorimetric method of
Standards, chemicals and solvents
Slinkard and Singleton,16 based on the oxidation of the hydroxyl
The commercially available standards of phenolic com- groups of phenols in basic media by the Folin–Ciocalteu reagent,
pounds – gallic acid, 5-O-caffeolyl-quinic acid and quercetin was used. Briefly, an aliquot (0.5 mL) of the extracts, blank or
3-O-rutinoside – were purchased from Sigma–Aldrich Chemie standard was placed in a 15 mL tube, where the Folin–Ciocalteu
GmbH (Taufkirchen, Germany). reagent (2.5 mL) was added and the mixture was allowed to react
The reagents 2,2-diphenyl-1-picrylhydrazyl radical (DPPH• ), 2,2′ - for 2 min under continuous stirring before a solution of sodium
azobis(2-methylpropionamidine) dihydrochloride (AAPH), carbonate (75 g L−1 , 2 mL) was added and mixed well. The mixed
monobasic sodium phosphate, dibasic sodium phosphate, was incubated at 50 ∘ C during 15 min. The absorbance was then
Folin–Ciocalteu reagent and fluorescein (free acid) were obtained measured at 750 nm using a Shimadzu UV-160A spectrophotome-
from Sigma–Aldrich, while 6-hydroxy-2,5,7,8-tetramethyl ter (Shimadzu, Kyoto, Japan). The results are expressed as mg gallic
chroman-2-carboxylic acid (Trolox) was purchased from Fluka acid equivalents (GAE) g−1 dried weight (DW), using a calibration
Chemika (Neu-Ulm, Switzerland). Meanwhile, formic acid, ethanol, curve over the range of 10–90 mg L−1 . This method is not spe-
methanol, hexane, acetone, dichloromethane and acetonitrile cific for polyphenols because other reducing compounds will also
were all of analytical grade (Merck, Darmstadt, Germany). The be included in the quantification, but it is widely used for stan-
chromatographic solvents were methanol, acetonitrile, ethyl dardization between studies and species and also for comparison
acetate (HPLC grade, procured from Merck). 𝛽-Carotene and lutein with other species. Therefore, we also used chromatographic and
were purchased from Sigma–Aldrich. Violaxanthin, 𝛼-carotene mass spectra analysis for the characterization of the polyphenols
and lutein were isolated from natural sources by classical chro- in guayusa samples.
matographic techniques.15

Identification of phenolic compounds by HPLC-DAD-ESI/MSn

Plant material and quantification by HPLC-DAD.
Leaves of fresh and processed guayusa (Ilex guayusa) were kindly The extraction and analysis of phenolic compounds were per-
provided by the RUNA Foundation (Archidona, Napo, Ecuador). formed using the method of Gironés-Vilaplana et al.17 In relation
Guayusa leaves were collected in Pastaza, Ecuador. The collection to the identification, it was carried out by HPLC-DAD-ESI/MSn anal-
3930

and the storage of the material were carried out under strictly yses, using an Agilent HPLC 1100 series model equipped with a

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Phytochemical composition of guayusa new tea www.soci.org

photodiode array detector and a mass detector in series (Agilent 2,2′ -azobis(2-methyl-propionamidine)-dihydrochloride
Technologies, Waldbronn, Germany). The equipment consisted of (250 mmol L−1 ) and antioxidant [Trolox (10–200 μmol L−1 ) or
a binary pump (model G1312A), an autosampler (model G1313A), guayusa samples (methanol/water, 70:30 v/v) (at different concen-
a degasser (model G1322A), and a photodiode array detector trations)]. DPPHA and ORAC assays were performed using 96-well
(model G1315B). The HPLC system was controlled by Chem-Station micro plates (Nunc, Roskilde, Denmark) and an Infinite® M200
software (Agilent, version 08.03). The mass detector was an ion microplate reader (Tecan, Grödig, Austria). Assays were conducted
trap spectrometer (model G2445A) equipped with an electro- in triplicate. The results are expressed as mmol Trolox 100 g−1 DW.
spray ionization interface, and was controlled by LCMSD software
(Agilent, version 4.1). The ionization conditions were 350 ∘ C and Statistical analysis
4 kV, for capillary temperature and voltage, respectively. The neb-
The data are presented are mean values (n = 3) ± standard devi-
ulizer pressure and nitrogen flow rate were 65.0 psi and 11 L min−1 ,
ation. The data were subjected to analysis of variance (ANOVA),
respectively. The full-scan mass covered the range of m/z from 100
with a 95% confidence level. Pearson’s correlation analysis was per-
to 1200. Collision-induced fragmentation experiments were per-
formed to corroborate the relationships between bioactive com-
formed in the ion trap using helium as the collision gas, with volt-
pounds (polyphenols and carotenoids) and antioxidant activity,
age ramping cycles from 0.3 to 2 V. The mass spectrometry data
with a 95% confidence level. The software used was Statgraphics
were acquired in the negative ionization mode. The MSn was car-
Centurion version 16.1.18 (Statgraphics.Net, Madrid, Spain).
ried out in the automatic mode on the more abundant fragment
ion in MS (n − 1).
For the quantification, the HPLC-DAD system (Agilent
1220-Infinity LC) was used and the compounds separated in a Luna
RESULTS AND DISCUSSION
Characterization of the phenolic composition
C18 column (25 cm × 0.46 cm, 5 μm particle size; Phenomenex,
Guayusa polyphenols were identified by HPLC-DAD-ESI-MSn
Macclesfield, UK). The individual phenolic compounds were tenta-
methodology (Table 1) and quantification of individual phenolic
tively identified following their characteristic UV–visible spectra,
compounds was carried out in HPLC-DAD. Besides, the traditional
order of elution [retention time (Rt)], and comparison with stan-
method of analysis of TPC was also carried out in the samples
dards previously established in the HPLC-DAD-ESI-MSn method.
using Folin–Ciocalteu reagent (Table 2).
Hydroxycinnamic acids were quantified using chlorogenic acid
With regard to the detection of individual phenolics from I.
(5-O-caffeolyl-quinic acid) as standard at 330 nm and flavonols
guayusa, a characteristic chromatogram for these samples is
using rutin (quercetin 3-O-rutinoside) at 360 nm. All the samples
shown in Fig. 1.
were extracted in triplicate and injected three times. The results
The characteristic UV–visible spectra, order of elution (Rt), par-
are expressed as mg g−1 DW.
ent ions (MS− ), along with the MS2 spectra provide confirmation to
discriminate between the different phenolic acids and flavonols.
Identification and quantification of carotenoid compounds by rapid A total of 14 phenolic compounds were detected of which nine
resolution liquid chromatography compounds corresponded to hydroxycinnamic acids and deriva-
The analyses of carotenoids were carried out according to the tives (peaks 1–7, 12 and 14) and five compounds were flavonoids,
method described by Stinco et al.10 The extracts were saponified specifically, flavonols (peaks 8–11 and 13). It is important to
to eliminate the chlorophylls. The saponification conditions were emphasize that the order of elution observed was the same as
methanolic KOH (30 g 100 mL−1 ) for 1 h under dim light and at previously described in Bravo et al.,20 with the exception of the
room temperature, after which they were washed with water to 3,4-dicaffeoylquinic acid, in yerba mate leaves.
remove any trace of base. The colored dichloromethane extracts
obtained were concentrated to dryness in a rotary evaporator at
Caffeoyl derivatives
temperature below 30 ∘ C and dissolved in 100 𝜇L of ethyl acetate
In relation to the caffeoyl quinic isomers detected, they are two
prior to their injection in the RRLC system. All the samples were
compounds (peaks 1 and 5) with similar mass spectra, with a [M]−
extracted in triplicate. The identification was made by comparison
ions at m/z 353 and 707 in the ESI− mode, and fragment ions at m/z
of their chromatographic and UV–visible spectroscopic character-
191 (base peak) and 179 (Table 1). The Rt and MSn spectra of com-
istics with those of standards. Meanwhile, the quantification was
pound 5 were identical to the standard chlorogenic acid, which
carried out by external calibration from the areas of the chromato-
corresponds to caffeic acid esterified to quinic acid (5-O-caffeoyl
graphic peaks obtained by DAD detection at 450 nm. The results
quinic acid). Fragment ions at m/z 191 (base peak) and a weak 179
are expressed as μg g−1 DW.
fragment (<5%), would correspond to deprotonated 5-O-caffeoyl
quinic acid and caffeic acid fragments, respectively, while m/z 707
Antioxidant capacity mass was a dimeric adduct of the caffeoylquinic acid molecules.
Antioxidant activity was determined by the DPPHA and ORAC The other compound, peak 1, would correspond to isomers of
methods adapted to a microscale. DPPHA test was realized chlorogenic acid, concretely 3-O-caffeoyl quinic acid (neochloro-
according to the method described by Mena et al.,18 where genic acid), where base peak m/z 191 and relatively intense sec-
the antioxidant activity was evaluated by measuring the vari- ondary ion at m/z 179 (∼50%), according to Clifford et al.21
ation in absorbance at 515 nm after 50 min of reaction with On the other hand, two compounds, 12 and 14, with a [M]−
DPPHA . The reaction was started by adding 2 𝜇L of the cor- ion at m/z 515 (Table 1) indicative of dicaffeoyl quinic acids, were
responding diluted sample to the well containing 250 𝜇L of also detected. Based on the MS and MS2 spectra and relative
DPPHA dissolved in methanol. In relation to ORAC, it was per- abundance, as well as the MS3 result of the base peak in the
formed according to Ou et al.19 Briefly, the reaction was carried MS2 fragmentation,21 were identified as 3,5-dicaffeoylquinic acid
out at 37 ∘ C in 10 mmol L−1 phosphate buffer (pH 7.4) and the (isochlorogenic acid) with MS2 at m/z 191, and 4,5-dicaffeoylquinic
3931

final assay mixture (200 𝜇L) contained fluorescein (1 μmol L−1 ), acid with MS2 at m/z 173, respectively (Table 1).

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 www.soci.org A García-Ruiz et al.

Table 1. Tentative identification of phenolics in guayusa samples by HPLC-DAD-ESI/MSn

Peak Rt (min) M− MS2 fragments Phenolic compounds

1 6.0 707*, 353 191 (100), 179 (42.6)** 3-O-CQA (neochlorogenic acid)
2 6.6 341 179 (63.0), 135 (10.6) Caffeoyl-hexose
3 8.2 341 179 (100), 135 (10.4) Caffeoyl-hexose
4 9.6 341 179 (78.9), 135 (4.9) Caffeoyl-hexose
5 10.6 353 191 (100), 179 (4.7) 5-O-CQA (chlorogenic acid)
6 12.5 707*, 353 191 (100), 179 (3.2) 5-O-CQA isomer
7 20.7 367 193 (51.3), 173 (0.8) 3-Feruloylquinic acid
8 27.3 609 301 (100), 271 (23.1) Quercetin-3-O-rutinoside (rutin)
9 28.5 609 301 (100), 271 (15.2) Rutin Isomer
10 32.5 463 301 (100) Quercetin-3-O-hexose
11 34.3 463 301 (100) Quercetin-3-O-hexose
12 36.1 515 353 (100), 191 (9.7)†, 179 (1.3) 3,5-Dicaffeoylquinic acid (isochlorogenic acid)
13 42.8 447 285 (100) Kaempferol-3-O-hexose
14 43.7 1031*, 515 353 (61.4), 173 (10.43)† 3,4-Dicaffeoylquinic acid

Rt, retention time.

* Dimeric adduct.
** Relative abundance (software ca. %).
† Base peak in MS2 (MS3 100% Rel. abun.).
CQA, caffeoylquinic acid.

Table 2. Phenolic composition in green leaves and processed guayusa

Concentration (mg g−1 DW)

Peak Compound Green leaves Blanched guayusa Fermented guayusa

Total phenolics content* 54.86b ± 3.09 106.62a ± 4.41 59.47b ± 1.15

Individual compounds
1 3-O-CQA (neochlorogenic acid) 7.93b ± 0.34 11.30a ± 1.06 2.55c ± 0.25
2 Caffeoyl-hexose 1.b ± 0.05 1.31a ± 0.14 0.12c ± 0.01
3 Caffeoyl-hexose 4.98a ± 0.73 5.17 a ± 0.17 1.29b ± 0.21
4 Caffeoyl-hexose 0.76b ± 0.09 1.71a ± 0.18 0.21c ± 0.10
5 5-O-CQA (chlorogenic acid) 24.10a ± 1.59 26.53a ± 0.84 7.53b ± 0.09
6 5-O-CQA†isomer < QL < QL < QL
7 3-Feruloylquinic acid 0.60a ± 0.05 0.58a ± 0.02 0.21b ± 0.01
8 Quercetin-3-O-rutinoside 0.60b ± 0.00 0.70a ± 0.02 0.32c ± 0.02
9 Quercetin-3-O-rutinoside 0.88b ± 0.02 0.96a ± 0.04 0.48c ± 0.02
10 Quercetin-3-O-hexose 2.64b ± 0.19 3.35a ± 0.33 1.54c ± 0.05
11 Quercetin-3-O-hexose 5.72b ± 0.66 5.96a ± 0.18 2.88b ± 0.17
12 3,5-Dicaffeoylquinic acid (isochlorogenic acid) 16.61b ± 0.20 23.34a ± 0.07 17.55b ± 1.25
13 Kaempferol-3-O-glucoside 1.02b ± 0.12 1.56a ± 0.12 0.79c ± 0.13
14 3,4-Dicaffeoylquinic acid 3.28b ± 0.05 4.74a ± 0.32 4.53a ± 0.60
* Total phenolics content, by the Slinkard and Singleton method, is expressed as mg of gallic acid equivalents per g.
a–c Mean values with different superscript letter in the same row indicate statistically significant differences among the three treatments (P < 0.05).
† CQA, Caffeoylquinic acid; QL, quantification limit.

Apart from the major peaks, other compounds were identified at m/z of 193 (51%) and 173 (0.8%). An ion at m/z 193 corresponds
as caffeoyl-hexoses (peaks 2, 3 and 4) as suggested from the to ferulic acid while an ion at m/z 173 is dehydrated quinic acid.
deprotonated molecular ion m/z of 341, the possible loss of caffeic
acid, m/z = 162 (can be confused with the loss of hexose), and Flavonols
confirmed by the MS2 base peak at m/z of 179 (>60%), with an
Five peaks (compounds 8–11 and 13) had MS spectra compatible
additional 135 m/z fragment.21
with a flavonols. As described with the compound 5, the Rt and
MS spectra of compounds 8 and 9 were identical to the standard
rutin, which corresponds to quercetin 3-O-rutinoside, showing a
Other hydroxycinnamoyl quinic isomer molecular ion m/z of 609 and fragments at m/z 301 (100%) and
A compound at 20.7 min (compound 7) showed the MS spectra of a 271. On the other hand, the peaks 10 and 11 were identified as
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3-feruloylquinic acid, with a mass of 367 and a molecular intact ion quercetin 3-O-hexose by presenting a [M]− ion at m/z 463 and

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Phytochemical composition of guayusa new tea www.soci.org

A
mAU
HPLC-DAD 360 nm
1000

800

600 9

400
8
200
6 7 11
0
B
1 4 HPLC-DAD 330 nm
3000

2500
10
2000

1500

1000
3
2 12
500
5
0
10 20 30 40 50 min

Figure 1. Typical chromatogram of guayusa, registered at 360 nm (A) and 330 nm (B) for the identification and quantification of phenolic compounds in
the samples. For compound assignment see Table 1.

characteristic fragment ions at m/z 301 (100%), according also flavonols was around 11 mg g−1 DW. This concentration is around
with order of elution showed by Schieber et al.22 for flavonols. 2, 20 and 28 times higher than the previously described for yerba
Finally, peak 13 had [M]− ion at m/z 447 and a fragment at m/z mate by Bravo et al.20 (around 5 mg g−1 DW), other Ilex spp. by Filip
285 (100%) in the negative mode, which is consistent with a et al.5 (around 0.5 mg g−1 ), and for tea by Peterson et al.29 (around
kaempferol-3-O-hexose. 0.4 mg g−1 DW), respectively. Quercetin-3-O-hexose was the most
The different compounds identified have been previously abundant flavonol glycoside. Biological studies suggested that
described as typical constituents of the phenolic fraction of the quercetin-3-O-hexose may have an antiviral, antibacterial, anticar-
leaves of teas,23 mate (I. paraguariensis)20,24 and other seven cinogenic and anti-inflammatory effects.30 Quercetin is also the
Ilex species (I. brevicuspis, I. theezans, I. microdonta, I. dumosa main representative of the flavonols in tea varieties.29
var. dumosa, I. taubertiana, I. pseudobuxus, I. integerrima and I. The industrial processing could modify the profile and concen-
argentina).5 tration of the phenolic fraction of guayusa. Thus, in the present
In relation to green leaves, quantitative analysis indicated that study also the polyphenol composition of guayusa was evaluated
the hydroxycinnamic acid derivatives were the major constituents after being subjected to industrial processes of blanching and fer-
of the phenolic fraction (85% of the total phenolic) (Table 2). Caf- menting. As shown in Table 2, both industrial processes did not
feic acid was the main cinnamate, with mono- and dicaffeoyl quinic modify the phenolic profile of guayusa. In relation with the con-
acid isomers accounting for approximately 75% and 90% of the centration of these compounds, the processed guayusa showed
total phenolic and hydroxycinnamic acids content, respectively. significant differences with respect to the green leaves, with an
Bravo et al.20 and Filip et al.5 also described the hydroxycinnamic increase in the content of polyphenols (except feruloyl-quinic acid)
acid isomers as the principal constituents of the phenolic fraction in the blanched guayusa and with a reduction in the content
of mate and seven Ilex species, respectively. Meanwhile, chloro- of phenolic compounds (except dicaffeoyl-quinic acid isomers)
genic acid with 24.10 mg g−1 DW stood out as the most abundant in the fermented guayusa (Table 2). During the blanching pro-
phenolic compound. This concentration is similar or higher to that cess, the temperature and humidity conditions as well as the fast
reported for the mate (21–28 mg g−1 )5,25 and for seven Ilex species cooling step would produce cell disruption which would lead to
(0.42–9.15 mg g−1 )5 and for tea (black tea = 0.5 mg g−1 ; green the subsequent release of polyphenols. This fact, together with
tea = 0.2 mg g−1 )26 but lower to that described for green coffee, the inactivation of enzymes involving phenolic oxidation, would
a major source of chlorogenic acid in nature (50–120 mg g−1 ).27 contribute to a more efficient extraction of polyphenols from the
Regarding their biological effects, numerous reports about chloro- blanched leaves and therefore, it would explain the high content of
genic acids focused on their antioxidant, anti-inflammatory, and polyphenols observed in the guayusa subjected to blanching. On
anti-obesity activities, cancer chemoprevention, and a reduction the other hand, polyphenol concentrations during fermentation
in the cardiometabolic problems, type 2 diabetes and Alzheimer’s may decrease due to their diffusion out of their storage cells and
disease.28 Chlorogenic acids are major constituents of plants, such further oxidation (non-enzymatic and/or catalyzed by polyphe-
as coffee beans28 , yerba mate24 and other Ilex spp.5 nol oxidase and peroxidase) and complexation into high molec-
In contrast, the flavonol fractions studied in tea and yerba mate, ular mass. The results obtained in the blanched guayusa are in
were described as rich in aglycones and glycosides.5 The flavonols line with the reported previously in processed and commercial
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in guayusa were glycosides (Table 2). The total concentration of mate by Isolabella et al.25 and Anesini et al.,31 respectively, while

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 www.soci.org A García-Ruiz et al.

Table 3. Carotenoid composition in green leaves and processed Table 4. Antioxidant capacity in green leaves and processed
guayusa guayusa

Concentration (μg g−1 DW) Antioxidant capacity

Blanched Fermented Assay Green leaves Blanched guayusa Fermented guayusa
Compound Green leaves guayusa guayusa
DPPH 33.0b ± 1.4 35.4a ± 1.0 19.7c ± 0.2
𝛼-Carotene 113.12a ± 19.81 62.52b ± 15.59 46.76b ± 2.00 ORAC 154.0a ± 20.4 111.3b ± 25.0 76.0c ± 5.3
𝛽-Carotene 37.85b ± 5.09 115.59a ± 27.46 58.34b ± 0.41 a–c Mean values with a different superscript letter in the same row indi-
Lutein 188.19ab ± 46.69 266.33a ± 65.38 173.38b ± 3.65 cate statistically significant differences among the three treatments
Violaxanthin + 108.46a ± 25.25 24.27b ± 2.06 8.17b ± 0.34 (P < 0.05).
neoxanthin Antioxidant capacity was determined as mmol Trolox 100 g−1 DW.
Total carotenoids 447.62ab ± 96.84 468.71a ± 110.49 286.65b ± 6.4
a,b Mean values with different letter on the right in the same row indi-
cate statistically significant differences among the three treatments
Table 5. Pearson’s correlation coefficients (r) between bioactive
(P < 0.05).
compounds (flavonoids and carotenoids) green leaves and processed
guayusa and its antioxidant capacity (DPPH and ORAC)

the results observed in fermented guayusa are agreed with the Samples
data described in black tea by Finger32 and Sang.33 On the other
Assay Green leaves Blanched guayusa Fermented guayusa
hand, as in green leaves, chlorogenic acid was the major phenolic
compound found in the blanched guayusa while in the fermented DPPH 0.73 0.98 0.80
guayusa 3,5-dicaffeoylquinic acid (isochlorogenic acid) was the ORAC 0.99 1.00 1.00
most abundant compound. The lowest 3,5-dicaffeoylquinic acid
content in the fermented guayusa could be due to the oxidation
(non-enzymatic and/or enzymatic) of this compound during fer- Therefore, it was evaluated how industrial processes, particu-
mentation process. larly blanching and fermentation, could modify its concentra-
Finally, green leaves showed a TPC of 54.86 mg GAE g−1 DW. This
tion. Thus, the blanched guayusa exhibited a higher content of
content is similar to that reported by Pardau4 in green guayusa
𝛽-carotene and lutein than green leaves (305.39 and 141.52%,
(54.92 mg GAE g−1 ), and higher to that reported by Moraes de
respectively), but lower concentration of 𝛼-carotene and violaxan-
Souza et al.34 in some processed tea and herbal infusions (ranging
thin + neoxanthin (55.27 and 22.38%, respectively) (Table 3). In this
0–46 mg GAE g−1 ), by Valerga et al.35 for fresh leaves of mate
sense, it appears that blanching of guayusa leaves is important to
(4.15 mg GAE g−1 ) and by Oh et al.36 for various leafy herbal teas.
retain the levels of these health-promoting carotenoids.
However, our results are lower to those described for green and
Similarly, to phenolic compounds, the fermented guayusa
black tea, water extracts (around 82 mg GAE g−1 ) by Oh et al.36
showed the lowest levels of carotenoids, except 𝛽-carotene
In relation to processed guayusa, blanching led to a significant
(Table 3). The fermented guayusa, exhibited lower contents of
increase (48.5%) in the TPC of guayusa in comparison to the green
𝛼-carotene and violaxanthin + neoxanthin than green leaves
leaves, but fermentation did not show significant differences with
(P < 0.05), but it did not show significant differences in the con-
the untreated leaves. The blanched guayusa showed the highest
centrations of 𝛽-carotene and lutein (Table 3). Silveira et al.40
TPC, being this content (107 mg GAE g−1 ) higher than observed in
described in the mate that the increase of the temperature of the
processed mate35 and green and black tea, water extracts.36
water for infusion favored the transfer of lutein. This could explain
the high content of lutein in the blanched guayusa, because this
Carotenoids industrial process proceeds at a high temperature water.
With regard to carotenoids, a total of five carotenoid compounds On the other hand, the major degradation of the carotenoids
were quantified in green leaves and guayusa processed: 𝛼- and 𝛼-carotene and violaxanthin + neoxanthin in the fermented
𝛽-carotene, lutein and violaxanthin + neoxanthin (Table 3). The guayusa could be attributed to the oxidative degradation
total content of carotenoids evaluated as the sum of the content of (enzymatic or non-enzymatic), principal cause of losses of
individual pigments did not show significant differences between carotenoids.37,39,42 In addition, the oxidative enzymes are inac-
green leaves and guayusa processed, differences were only signif- tivated during blanching42 which would explain that despite of
icant for blanched and fermented guayusa (Table 3). The total of the heat treatment employed in this process, the destruction
carotenoids ranged from 286.65 to 468.71 μg g−1 DW, which was of 𝛼-carotene and violaxanthin + neoxanthin was lower in the
in good agreement with the results reported by Ravichandran37 in blanched guayusa than in fermented guayusa. With regard to
tea leaves. On the other hand, lutein was the main carotenoid in violaxanthin + neoxanthin, it was the most labile carotenoid to
the green leaves, which agreed with the results reported by other the industrial treatments, with percentages of degradation of the
authors in green leafy vegetables.38,39 This carotenoid plays impor- 77.6% and 92.5% in the blanched guayusa and fermented guayusa,
tant roles in human health, such as the reduction of risk of diseases respectively. These results are in accordance with the results
as cancer, cataracts and age related macular degeneration.39 – 41 reported by other authors in green vegetables.39,43 The changes
The contents of 𝛽-carotene and lutein were in the range reported observed in the content of carotenoid during guayusa processing
in teas37,38,41 while the concentrations of 𝛼-carotene and violaxan- could origin the production of various carotenoid-derived aroma
thin were higher as compared to other reported data.37 compounds that would contribute positively to the organoleptic
Being highly unsaturated, carotenoids are susceptible to modi- characteristic of guayusa, which was in agreement with that
3934

fications (i.e. isomerization and oxidation) during processing.39,42 described by other authors in the manufactured tea.37,44

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Phytochemical composition of guayusa new tea www.soci.org

Antioxidant capacity Thematic Networks). A.G.R. also thanks to the Ecuadorian Secre-
Numerous studies have highlighted the antioxidant capacity of tary of Higher Education, Science, Technology and Innovation for
polyphenols and carotenoids.7,39 Antioxidant properties of the the Prometeo Postdoctoral Grant. N.B. was funded by the FPU Fel-
green leaves and processed guayusa were evaluated on the lowship Program of the Spanish Ministry of Education. The authors
basis of measuring scavenging activity for DPPH radicals and the would like to thank RUNA Foundation for the samples supplied.
inhibition of peroxyl radical induced oxidations by antioxidants
by ORAC method. The results for the antioxidant capacity are
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