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Phenolic compounds, antioxidant capacity, and in

vitro α-amylase inhibitory potential of tea infusions
(Camellia sinensis) commercialized in Chile
a a b a
Ana María Quesille-Villalobos , Jorge Saavedra Torrico & Lena Gálvez Ranilla
a
Escuela de Alimentos, Facultad de Recursos Naturales, Pontificia Universidad Católica
de Valparaíso , Avenida Waddington 716, Playa Ancha , Valparaíso , Chile
b
Centro Regional de Estudios en Alimentos Saludables (CREAS) , Valparaíso , Chile
Published online: 31 May 2012.

To cite this article: Ana María Quesille-Villalobos , Jorge Saavedra Torrico & Lena Gálvez Ranilla (2013) Phenolic
compounds, antioxidant capacity, and in vitro α-amylase inhibitory potential of tea infusions (Camellia sinensis)
commercialized in Chile, CyTA - Journal of Food, 11:1, 60-67, DOI: 10.1080/19476337.2012.688219

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 CyTA – Journal of Food
Vol. 11, No. 1, February 2013, 60–67

Phenolic compounds, antioxidant capacity, and in vitro a -amylase inhibitory potential of tea
infusions (Camellia sinensis) commercialized in Chile
Compuestos fenólicos, capacidad antioxidante y potencial inhibitorio in vitro de a -amilasa en
infusiones de té (Camellia sinensis) comercializados en Chile
Ana Marı́a Quesille-Villalobosa, Jorge Saavedra Torricoa,b and Lena Gálvez Ranillaa*
a
Escuela de Alimentos, Facultad de Recursos Naturales, Pontificia Universidad Católica de Valparaı´so, Avenida Waddington 716,
Playa Ancha, Valparaı´so, Chile; bCentro Regional de Estudios en Alimentos Saludables (CREAS), Valparaı´so, Chile
(Received 31 December 2011; final version received 22 April 2012)

Chile is considered the largest tea consumer in America, so a broad variety of tea types and brands are available in Chilean markets.
Downloaded by [University of Dayton] at 21:25 28 December 2014

Thirty-four commercial teas (white, green, red, oolong, and black) commonly found in Chile were evaluated according to their
phenolic profiles, antioxidant capacity, and in vitro inhibition against a-amylase relevant for hyperglycemia control. Multivariate
tools were used to evidence patterns among samples respect their biochemical parameters. Green and white tea groups showed
higher total phenolic contents than red and black teas. All tea samples inhibited the 2,2-diphenyl-1-picrylhydrazyl free radical and
black teas significantly inhibited the a-amylase enzyme. Major phenolics detected by liquid chromatography in green and white teas
were epigallocatechingallate and epicatechingallate, whereas gallic acid was common in black, red, and oolong teas. Caffeine was
found across all samples. The multivariate analysis evidenced a high variability among analyzed tea and even from brand to brand.
Keywords: Camellia sinensis; phenolics; free radical inhibition; a-amylase inhibition

Chile es considerado el mayor consumidor de té en América y una amplia variedad en tipos y marcas de té existen en el mercado
chileno. Un total de treinta y cuatro muestras de té (blanco, verde, rojo, oolong y negro) fueron evaluadas de acuerdo a sus perfiles
de compuestos fenólicos, capacidad antioxidante e inhibición in vitro de la a-amilasa, relevante en el control de la hiperglicemia. Se
utilizaron herramientas multivariantes para evidenciar patrones entre las muestras respecto a sus parámetros bioquı́micos. Los
grupos de té verdes y blancos presentaron los valores más altos en fenólicos totales. Todas las muestras inhibieron el radical libre
2,2-difenil-1-picrilhidracilo, en cambio sólo los tés negros inhibieron significativamente la enzima a-amilasa. Los principales
compuestos fenólicos detectados por cromatografı́a lı́quida en té verde y blanco fueron la epigalocatequina-galato y epicatequina-
galato, mientras que ácido gálico fue detectado en las muestras de té negro, rojo y oolong. Se detectó cafeı́na en todas las muestras
evaluadas. El análisis multivariante demostró una alta variabilidad entre las muestras de té y aún entre las diferentes marcas
procedentes de un mismo tipo de té.
Palabras claves: Camellia sinensis; fenólicos; inhibición de radicales libres; inhibición a-amilasa

Introduction Sri Lanka (http://www.odepa.gob.cl). Although all tea types

Tea is the most popular drink after water worldwide and is
made from leaves of Camellia sinensis (Theaceae family), a
plant native from the southeastern China and cultivated
across the world mainly in tropical and subtropical areas
(Sultana et al., 2008). Several countries have adopted tea as a
traditional drink in spite of the fact that many of them are
not considered tea producers. This is the case of Chile, where
tea consumption is significantly high and plays an important
role on Chilean culture. Recent data show that tea intake in
Chile is around 319.5 cups per capita, the largest among
American countries, only followed by the United States,
Colombia, and Brazil (150.2, 11.3, and 9.1 cups, respectively)
(http://www.oficinascomerciales.es/icex/cda/controller/page
Ofecomes/0,5310,5280449_5282927_5284940_4237341_CL,0
0.html). This situation explains the large amounts of tea
importations, especially black and semi-fermented teas
(17,780.8 tons in 2010) coming mainly from Argentina and
arise from the same plant (C. sinensis), differences in tea
chemical composition are mainly related to their manufacture
conditions, growth conditions, and area of cultivation. Based
on processing and the harvested leaf development, tea types
are classified into: white (harvested leave buds with white
trichomes, non-fermented or semi-fermented), green teas
(non-fermented), oolong teas (semi-fermented), and black
teas which are fully fermented. Pu-erh or red teas are
postfermented teas as opposed to fermented teas, such as
black tea (Gonzalez de Mejia, Ramirez, & Puangpraphant,
2009; Ku, Kim, Park, Liu, & Lee, 2010). Phenolic
compounds present in fresh tea leaves, mainly flavanol
monomers and gallates, go through important chemical
changes during the fermentation process. Thus, catechins are
enzymatically oxidized or condensed to a wide array of
complex products, such as the polymers theaflavins and
thearubigins (Kim, Goodner, Park, Choi, & Talcott, 2011).

*Corresponding author. Email: lena.galvez@ucv.cl

ISSN 1947-6337 print/ISSN 1947-6345 online

Ó 2013 Taylor & Francis
http://dx.doi.org/10.1080/19476337.2012.688219
http://www.tandfonline.com
 CyTA – Journal of Food 61

These phenolic compounds are responsible for certain black Table 1. Sample description.
tea characteristics, such as color and taste (Gonzalez de Tabla 1. Descripción de muestras.
Mejia et al., 2009).
Labeled
Besides the cultural aspect, the overall increase in tea
Group No. Commercial name origin pH*
consumption may also be due to the numerous studies which
have shown evidence about the potential health benefits Oolong 1 Dilmah Oolong Sri Lanka 5.5 + 0.1
linked to tea. Tea phenolic compounds, such as catechins are Red 2 Twinings Pu-erh China 5.4 + 0.1
3 Dilmah Pu-erh Sri Lanka 5.6 + 0.2
thought to play a role on preventing or reducing the risk of 4 Butterfly Pu-erh China 5.7 + 0.2
oxidation-linked chronic diseases, such as diabetes, cardio- White 5 Butterfly White China 5.5 + 0.2
vascular diseases, and likely cancer according to recent 6 Twinings White China 5.6 + 0.2
studies (González-Manzano et al., 2011; Tabassum, Siddiqui, 7 Supremo White China 5.7 + 0.1
& Rizvi, 2010). The antioxidant properties of tea phenolic Green 8 Akbar Green Sri Lanka 5.6 + 0.1
9 Dilmah Sencha Green Sri Lanka 5.6 + 0.1
compounds have also been documented and different 10 Teekanne Green Not informed 5.5 + 0.1
mechanisms have been proposed (Frei & Higdon, 2003). 11 Butterfly Green China 5.9 + 0.1
Further, the inhibitory action of tea polyphenols against 12 Supremo Green China 5.7 + 0.1
mammalian a-amylases has been reported (Koh, Wong, Loo, 13 Lipton Green Not informed 5.8 + 0.1
Black 14 Akbar Eng. Breakfast Sri Lanka 5.0 + 0.1
Kasapis, & Huang, 2010). This property could lead to the 15 Teekanne Gold Sri Lanka 5.0 + 0.0
reduction of starch hydrolysis potentially reducing the 16 Lipton Royal Sri Lanka 5.0 + 0.0
postprandial hyperglycemia. Therefore, tea could be con- 17 Supremo Express Sri Lanka 5.1 + 0.0
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sidered a healthy drink with bioactive molecules and high 18 Dilmah Ceylon Sri Lanka 5.1 + 0.1
antioxidant capacity (Gonzalez de Mejia et al., 2009). 19 Akbar Ceylon Sri Lanka 5.1 + 0.1
20 Lipton Yellow Label Kenya 5.1 + 0.0
A wide variety of tea types and brands are available in 21 Club Red Label Argentina 5.2 + 0.0
Chilean markets, however, few studies exist about their 22 Club Ceylon Sri Lanka 5.1 + 0.1
chemical composition and potential health-linked properties. 23 Emblem Not informed 5.1 + 0.1
Such information would be helpful to consumers who are 24 Mildred Sri Lanka 5.1 + 0.1
25 Supremo Brazil Brazil 5.1 + 0.0
continuously looking for healthier food and beverage 26 Club Seleccion Sri Lanka 5.3 + 0.1
choices, especially in Chile where the emergence of obesity- 27 Club Green Label Brazil 5.0 + 0.1
linked chronic diseases is reaching high rates as in developed 28 Twinings Darjeeling China 5.0 + 0.1
countries (Albala, Vio, Kain, & Uauy, 2002). Also, informa- 29 Twinings English Breakfast China 5.0 + 0.2
30 Twinings Lapsang Souchang China 5.2 + 0.1
tion based on biochemical parameters would allow the
31 Supremo Golden Sri Lanka 5.1 + 0.1
evaluation of the overall quality of tea products available 32 Supremo Premium Sri Lanka 5.0 + 0.1
for Chilean consumers. From above rationale, the objective 33 Aroma Sri Lanka 5.3 + 0.2
of this study was to characterize and compare 34 tea products 34 Superior Sri Lanka 5.2 + 0.1
(water infusions from black, red, green, oolong, and white Note: *Aqueous tea infusions at 258C.
teas) commercialized in Chile respect their chemical composi-
tion (phenolic profiles) and some functional properties, such
as antioxidant capacity and a-amylase inhibitory activity
using in vitro models. (7)-epigallocatechingallate (EGCG), (7)-epicatechingallate
(ECG), and caffeine (CA)] were purchased from Sigma
Chemical Co. (St Louis, MO, USA). Unless noted, all
Materials and methods chemicals were HPLC grade for the HPLC analysis.

Samples and infusion preparation

Different tea types (black, green, oolong, red, and white) of
34 commercially available bagged teas (Camellia sinensis)
were purchased from a local supermarket in Valparaiso,
Chile. The characteristics of analyzed samples are summar-
ized in Table 1.
Tea infusions were prepared following the traditional
home preparation: 2 g of tea were mixed with 200 mL of
distilled water at 1008C and soaked for 10 min under
agitation. Next, tea extracts were centrifuged for about
15 min at 6037 6 g and the supernatant was recovered and
stored at 558C until analysis. For each commercial tea
sample, three bags were analyzed and the extraction
procedure was run in triplicate.
Total phenolics
The analysis was performed according to Shetty, Curtis,
Levin, Wikowsky, and Ang (1995). Briefly, 0.5 mL of tea
extract was transferred into a test tube and mixed with 1 mL
of 95% ethanol and 5 mL of distilled water. To each sample,
0.5 mL of Folin–Ciocalteu (1N) was added and mixed. After
5 min at room temperature, 1 mL of sodium carbonate
(Na2CO3) solution was added to the reaction mixture and
allowed to stand for 1 h in a dark place. The absorbance was
read at 725 nm. The standard curve was built using various
concentrations of GA in 95% ethanol, and the results were
expressed as mg of gallic acid equivalents (GAE) per g of
sample weight.

Chemicals Antioxidant capacity by the DPPH radical inhibition

Porcine pancreatic a-amylase (EC 3.2.1.1), the 2,2-diphenyl- assay
1-picrylhydrazyl (DPPH) radical, the Folin–Ciocalteu re- The antioxidant capacity was determined by the DPPH
agent, and the pure HPLC standards [gallic acid (GA), radical scavenging method by an assay modified by Kwon,
 62 A.M. Quesille-Villalobos et al.
Vattem, and Shetty (2006). To 1 mL of 60 mM DPPH in 95%
methanol, 200 mL of each tea extract was added, and the
decrease in absorbance was measured after 1 min at 517 nm.
A control (distilled water instead tea extract) was also
included. The percentage of inhibition was calculated
according to the Equation (1):
A517 control  A517 extract
%Inhibiton ¼  100: ð1Þ
A517 control
a-Amylase inhibition assay
The a-amylase inhibitory activity was determined by an assay
modified from the Worthington Biochemical Corp. (1993). A
total of 500 mL of each sample extract and 500 mL of 0.02 M
sodium phosphate buffer (pH 6.9 with 0.006 M NaCl)
containing a-amylase solution (0.5 mg/mL) were incubated
at 258C for 10 min. After preincubation, 500 mL of a 1%
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starch solution in 0.02 M sodium phosphate buffer (pH 6.9
with 0.006 M NaCl) was added to each tube at timed
intervals. The reaction mixtures were then incubated at 258C
for 10 min. The reaction was stopped with 1.0 mL of
dinitrosalicylic acid color reagent. The test tubes were then
incubated in a boiling water bath for 10 min and cooled to
room temperature. The reaction mixture was then diluted
after adding 15 mL of distilled water, and absorbance was
measured at 540 nm. The absorbance of sample blanks
(buffer instead of enzyme solution) and a control (buffer in
place of sample extract) were recorded as well. The final
extract absorbance (A540 extract) was obtained by subtract-
ing its corresponding sample blank reading. The a-amylase
inhibitory activity was calculated according to the Equation
(2), as follows:
A540 control  A540 extract
%Inhibiton ¼  100: ð2Þ
A540 control
High performance liquid chromatography analysis
Tea infusions were filtered (pore size 0.2 mm) and the HPLC
analysis was performed according to Zuo, Chen, and Deng
(2002) with some modifications as follows. A HPLC system
series 200 with UV/Vis detector (PerkinElmer Inc., Shelton,
CT, USA) equipped with a binary pump, an autosampler and
controlled by the TotalChrom software (Perkin Elmer Inc.,
Shelton, CT, USA) was used. The analytical column was a
HibarLiChrospher 100 RP-18 (5 mm) (Merck, Darmstadt,
Germany). The injection volume was 10 mL, the flow rate was
0.5 mL/min, and the eluates were monitored at 280 nm. The
solvents used for gradient elution were: A – water/acetic acid
(97:3) and B – 100% methanol. The methanol concentration
was increased to 40% over the first 6 min, then to 60% for
the next 16 min, then to 70% within the next 2 min, and to
100% over the next 2 min. Finally, the gradient was changed
to initial conditions and maintained for 4 min (total run time
30 min). Phenolic compounds were identified using pure
HPLC standards based on their retention times. Co-
chromatography was used when necessary. Concentrations
of GA, EGCG, ECG, and CA were determined according to
their calibration curves (peak area versus concentration)
obtained for each compound (R2  0.999). The results were
expressed in mg/g of tea.
Statistical analysis
Extractions were run in triplicate and all analyses were
carried out in triplicate. Results were expressed as mean +
standard deviation. Data were subjected to one-way analysis
of variance (ANOVA) with the HSD Tukey test for multiple
comparisons. Variance normality and homogeneity were
evaluated with the Kolmogorov–Smirnov and Levene test
(Montgomery, 2001). The significance level was a ¼ 0.05. All
the univariate statistics were performed using the Stat-
graphics Centurion XVI (StatPoint Inc., Rockville, MD,
USA). Multivariate analysis was performed by principal
component analysis (PCA) and the partial least square
discriminant analysis (PLS-DA) according to Saavedra
et al. (2011), using SIMCA-P þ 12 (Umetrics AB, Umea,
Sweden). Data were centered and scaled to variance one prior
to the multivariate analysis. All analyses were validated by a
full cross-validation routine to minimize the predicted
residual sum of squares function thus avoiding model over
fitting (Cen, He, & Huang, 2007).
Results and discussion
Total phenolic contents
According to Table 2, a high variability was observed in the
total phenolic contents among all analyzed tea types and
brands (p 5 0.05). Total phenolics ranged from 37 + 1 to
112 + 0.1 mg GAE/g. The Twinings white tea and the Akbar
green tea showed the highest values among all samples
(112 + 0.1 and 111 + 1 mg GAE/g, respectively), whereas
the Twinings Lapsang Souchang black tea had the lowest level
(37 + 1 mg GAE/g). Overall, white and green tea infusions
showed higher total phenolic contents than the black tea
group with the exception of the Twinings Darjeeling black tea
which had a different trend (104 + 4 mg GAE/g). Hilal and
Engelhardt (2007) pointed out that, in general, black tea from
Darjeeling may contain up to 10% more catechins than some
green teas.
Difference in the total phenolic contents in tea samples
can be influenced by several factors, such as the diversity of
used raw material (sinensis or assamic ecotypes), the
geographic origin, type of processing (fermented or non-
fermented), and the extraction conditions for their analysis.
Considering the effect of manufacture conditions, it is well
known that during the fermentation process, the quantity of
simple catechins, commonly found at high levels in non-
fermented teas, decreases due to oxidation reactions cata-
lyzed by natural polyphenol oxidases. These reactions lead to
the increase of larger phenolic molecules, such as theaflavins
and thearubigins in black teas (Ankolekar et al., 2011). In
this way, Deetae, Parichanon, Trakunleewatthana, Chansee-
tis, and Lertsir (2012) found that total catechin contents were
significantly higher in green teas than in oolong (semi-
fermented) and black teas (fermented). Also, other studies
revealed that green teas have more total phenolic compounds
than black teas (Moraes de Souza, Oldoni, Reginato, &
Alencar, 2008).
Anesini, Ferraro, and Filip (2008) reported that total
polyphenol concentration was higher in green teas (non-
fermented) than in black teas (fermented) from Argentina.
Levels varied from 211 to 143 mg GAE/g in green teas, and
from 176 to 84 mg GAE/g in black teas. These ranges are
 CyTA – Journal of Food 63

Table 2. a-Amylase inhibitory activities, DPPH radical scavenging DPPH free radical scavenging capacity than red and black
capacity, and total phenolic contents in tea infusions. teas. However, variability among samples was lower when
Tabla 2. Actividad inhibidora de a-amilasa, capacidad de barrido compared to that observed among the total phenolic results.
de radical DPPH y contenidos totales de fenólicos en infusiones de té. White teas, such as the Butterfly white and Supremo white
a-Amylase Total showed a 64 + 1% and 64 + 2% of inhibition, respectively.
inhibitory Antioxidant phenolics In case of the green tea group, the Dilmah Sencha green tea
Sample activity (%) capacity (%) (mg GAE/g) showed the highest value (64 + 3%) and the Dilmah oolong
1 4+ 5 63 + 2 67 + 4 tea (semi-fermented) significantly inhibited the DPPH radical
2 3+ 4 49 + 3 53 + 7 (63 + 2%). In contrast, the lowest DPPH inhibition was
3 3+ 3 53 + 1 52 + 7 found in the Twinings Pu-erh red tea (49 + 3%) and in the
4 3+ 4 54 + 3 47 + 6 Twinings Darjeeling (50 + 6%) and Club Seleccion
5 17 + 2 64 + 1 78 + 4 (49 + 7%) black teas. Results from current work are
6 19 + 1 61 + 1 112.2 + 0.1
7 3+ 4 64 + 2 71 + 5 consistent with other studies where green teas also showed
8 30 + 3 57 + 1 111 + 1 the highest free radical scavenging capacities, closely followed
9 ND 64 + 3 65 + 1 by black teas (Deetae et al., 2012).
10 0.1 + 0.3 63 + 6 88 + 1 Several factors, such as those influencing the total phenolic
11 2+ 3 54 + 3 72 + 4
12 1+ 2 54 + 3 65 + 4 contents may explain variability observed in analyzed samples.
13 21 + 2 57 + 3 88 + 3 As mentioned above, the oxidation degree of tea leaves
14 54 + 2 55 + 1 60 + 5 determines the characteristic phenolic profiles and this likely
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15 56 + 4 58 + 3 66 + 4 influences the free radical scavenging-linked antioxidant

16 80 + 2 57 + 1 67 + 5 properties in teas. Non-fermented teas are generally rich in
17 46 + 6 55 + 1 68 + 7
18 57 + 4 60 + 3 67 + 8 flavonoids, such as catechin (C), epicatechin (EC), epigalloca-
19 56 + 3 60 + 4 67 + 6 techin (EGC), epicatechingallate (ECG) and epigallocatechin-
20 37 + 4 59 + 1 65 + 5 gallate (EGCG) (Gonzalez de Mejia et al., 2009). Karori,
21 0.5 + 1 57 + 1 55 + 2 Washira, Wanyoko, and Ngure (2007) found that high levels of
22 53 + 2 52 + 1 65 + 2
23 46 + 4 56 + 3 72 + 10
EGCG, EC, EGC, C, and ECG in green tea extracts strongly
24 51 + 2 56 + 1 69 + 6 correlated to high DPPH radical inhibition. The presence of a
25 21 + 2 57 + 2 64 + 4 trihydroxyphenyl B-ring in EGCG and EGC would explain the
26 36 + 4 49 + 7 65 + 2 potent free radical scavenging properties of such gallocatechins
27 23 + 3 53 + 3 62 + 4
(Shang et al., 2002).
28 44 + 6 50 + 6 104 + 4
29 21 + 3 52 + 3 63 + 5 Black teas also showed the capacity to inhibit the DPPH
30 12 + 1 51 + 3 37 + 1 free radical though in a lesser extent than white teas.
31 37 + 5 50 + 8 66 + 5 Polymeric theaflavins found in fermented teas contain
32 38 + 3 51 + 8 65 + 5 additional hydroxyl groups which make them able to donate
33 32 + 4 51 + 4 62 + 6
34 52 + 5 52 + 4 68 + 3 protons due to resonance delocalization. Further, GA is
another common phenolic compound detected in black teas
Notes: Results are expressed as means + SD. ND, not detected. and may also be associated to their antioxidant activities
Notas: Los resultados se muestran como media + DS. ND, no detectado. (Karori et al., 2007).

a-Amylase inhibitory potential

higher than values obtained for green and black teas in
current study and may be related to the fact that above
authors used methanol for phenolics extraction. The analysis
of water soluble phenolic compounds in tea infusions would
give more physiologically relevant results for overall potential
phenolic-linked health benefits.
Some Pu-erh red teas are produced by ripening unox-
idized tea leaves for several months using some yeast species
prior to being pressed for further postfermentation process.
Previous studies have shown that microbes in Pu-erh teas
may grow at the expense of several benzene compounds (Ku
et al., 2010). This fact could also explain why analyzed red
teas showed overall low total phenolic contents (from 47 + 6
to 53 + 7 mg GAE/g) when compared to non-fermented teas
and black teas in this study.
2,2-Diphenyl-1-picryhydrazyl (DPPH) radical
scavenging capacity
The antioxidant capacities of analyzed samples are shown in
Table 2. In general, white and green teas exhibited higher
The control of postprandial hyperglycemia is critical in the
early therapy for Type 2 diabetes. One therapeutic approach
to decrease hyperglycemia is to delay glucose absorption
through the inhibition of carbohydrate-hydrolyzing enzymes,
such as a-amylases and a-glucosidases at intestinal level
(Ankolekar et al., 2011; Chen, Qu, Fu, Dong, & Zhang,
2009). Green, black, and oolong teas have shown potential
antidiabetic properties and therefore may play a role on
lowering blood glucose and preventing hyperglycemia
(Buyukbalci & El, 2008).
The in vitro inhibition of porcine pancreatic a-amylase
significantly varied among analyzed samples (Table 2).
Almost all black teas inhibited the a-amylase, and the
inhibitory activities ranged from 12 + 1% to 80 + 2%.
The Lipton Royal black tea had 80 + 2% of inhibition,
followed by Dilmah Ceylon (57 + 4%), Teekanne Gold
(56 + 4%), and Akbar Ceylon (56 + 3%). A different trend
was shown by the Club Red Label black tea from Argentina,
since no inhibitory activity against a-amylase was observed.
The a-amylase inhibition was not significant in case of all red
teas, the Supremo white tea, and most of the green teas. The
Akbar green tea, which also showed a high total phenolic
 64 A.M. Quesille-Villalobos et al.

content (111 + 1 mg GAE/g), moderately inhibited the a- Table 3. Compounds detected at 280 nm by HPLC in tea infusions.
amylase (30% + 3).
Tabla 3. Componentes detectados a 280 nm por HPLC (Cromato-
Polymeric phenolic compounds, such as theaflavins and grafı́a lı́quida de alta eficacia) en infusiones de té.
thearubigins have been associated to the anti-hyperglycemia
properties observed in black teas. Previous in vitro and in vivo Content (mg/g)
studies have shown that isolated theaflavins have higher Gallic Epigalloca
a-amylase inhibitory activities than catechins due to their Sample acid Epicatechingallate techingallate Caffeine
affinity for protein-like enzymes (Honda, Nanjo, & Hara,
1 2 +1 7+1 31 + 2 26 + 2
1994). Koh et al. (2010) reported that less quantity of black 2 10 +1 ND ND 41 + 3
tea is necessary to inhibit the a-amylase and a-glucosidase 3 8 +1 ND ND 37 + 4
enzymes than in case of green teas and also Ankolekar et al. 4 2.5 + 0.5 ND ND 31 + 3
(2011) found that fully fermented teas had the highest 5 ND 13 + 1 30 + 2 31 + 2
a-amylase inhibitory activities (84.1–71.6%). Also, McDou- 6 ND 35 + 5 60 + 9 32 + 4
7 ND 14 + 1 37 + 3 19 + 0.4
gall et al. (2005) found that fruits rich in tannin compounds 8 ND 39 + 3 56 + 4 32 + 2
exhibited high a-amylase inhibitory properties than green 9 ND 9+2 35 + 5 19 + 1
teas (poor in tannin compounds). This rationale would 10 ND 13 + 1 49 + 3 25 + 1
explain why overall all black teas (fermented) strongly 11 ND 11 + 1 38 + 1 23 + 1
12 ND 9+1 38 + 4 17 + 1
inhibited the a-amylase among all analyzed samples in this 13 ND 16 + 1 48 + 4 24 + 1
study. 14 4+1 7+1 ND 29 + 0.6
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Koh et al. (2010) indicated that tannin’s inhibitory action 15 4.1 + 0.4 3 + 0.4 ND 28 + 2
would be attributed to non-covalent interactions, hydrogen 16 3+1 7 + 0.5 ND 26 + 1
bonding, and p–p interactions between the tannin aromatic 17 4.6 + 0.4 8+1 ND 34 + 2
18 4+1 8+1 ND 26 + 2
ring and proteins. 19 4 + 0.3 8+1 ND 27 + 1
20 4+1 7+1 ND 29 + 2
21 2.4 + 0.2 ND ND 21 + 1
HPLC analysis of phenolic compounds and CA 22 3 + 0.5 ND ND 25 + 1
23 4 + 0.5 8 + 0.5 ND 32 + 1
Specific phenolics and CA contents detected by HPLC are 24 5+1 8+1 ND 32 + 2
shown in Table 3, and Figure 1 shows some characteristic 25 3 + 0.4 ND ND 21 + 2
HPLC profiles for green, red, black, and oolong tea 26 4.3 + 0.3 6+1 ND 31 + 1
infusions. 27 3 + 0.5 5.6 + 0.4 ND 24 + 1
Significant differences were observed among phenolic 28 ND 22 + 2 42 + 3 32 + 2
29 2 + 0.4 6+1 ND 26 + 1
profiles and were likely related to the type of tea and brand. 30 1.1 + 0.1 ND ND 24 + 3
Overall, EGCG and ECG were the major phenolic com- 31 4.2 + 0.1 7 + 0.4 ND 29 + 1
pounds detected in white and green teas. Among green teas, 32 4.2 + 0.5 7 + 0.4 ND 29 + 1
contents of EGCG and ECG ranged from 35 + 5 to 56 + 4 33 3.2 + 0.3 ND ND 25 + 2
34 2.7 + 0.6 ND ND 26 + 4
mg/g and from 9 + 1 to 39 + 3 mg/g, respectively. In case
of white teas, the levels of EGCG and ECG varied from 30 to Notes: Results are expressed as means + SD. ND, not detected.
60 mg/g and from 13 to 35 mg/g, respectively. The Twinings Notas: Los resultados se muestran como media + DS. ND, no detectado.

Figure 1. HPLC chromatograms of tea infusions at 280 nm. (a) Oolong tea (Dilmah), (b) green tea (Akbar), (c) black tea (Twining Lapsang
Souchang) and (d) pu-erh red tea (Dilmah). GA, gallic acid; EGCG, epigallocatechingallate; ECG, epicatechingallate; CA, caffeine.
Figura 1. HPLC de infusiones de té a 280 nm. (a) té oolong (Dilmah), (b) té verde (Akbar), (c) té negro (Twining Lapsang Souchang) y (d) té rojo
Pu-erh (Dilmah). GA, ácido gálico; EGCG, epigalocatequina galata; ECG, epicatequina galata; CA, cafeı́na.

white tea showed the highest EGCG content (60 + 9 mg/g),
whereas the Akbar green tea was remarkable due to its high
concentration of ECG (39 + 3 mg/g) and EGCG (56 + 4
mg/g). Similar concentrations were found by previous
studies. Rusak, Komes, Likic, Horzic, and Kovac (2008)
and Cheong, Park, Kang, Ko, and Seo (2005) reported that
contents of EGCG and ECG in green tea infusions ranged
from 51.5 to 88.3 mg/g and from 7.5 to 18.9 mg/g,
respectively. Further, contents of 65 and 14 mg/g for
EGCG and ECG, respectively, were also reported in white
tea infusions (Rusak et al., 2008).
Major phenolic compounds found in black teas were GA
and ECG, and concentrations ranged from 1.1 + 0.1 to
5 + 1 mg/g and from 3 + 0.4 to 22 + 2 mg/g, respectively.
No EGCG was detected in black teas with exception of the
Twinings Darjeeling black tea (42 + 3 mg/g). Similarly, a
different trend related to its total phenolic content was
observed in this brand as mentioned above. Gallic acid,
ECG, and EGCG were simultaneously found in the semi-
Downloaded by [University of Dayton] at 21:25 28 December 2014
fermented Dilmah oolong tea, thus showing a different
phenolic profile among all tea types. Trihydroxylflavan-3-
ols, such as EGCG and EGC are generally oxidized faster
during the fermentation phase of black tea processing and
GA is most likely produced from the degradation of EGCG
(Lee et al., 2008). This would explain differences observed in
the HPLC phenolic profiles of analyzed tea types in current
work; however, the effect of the brand was also important.
The non-phenolic compound CA was found in all
samples (Table 3). The Twinings Pu-erh red tea had the
highest CA content (41 + 3 mg/g), whereas the Supremo
green tea showed the lowest amount (17 + 1 mg/g). In black
teas, variability among CA concentrations was lower than
that observed in white, green, and red teas. Previous studies
reported similar CA levels ranging from 29.2 to 56.3 mg/g in
green tea infusions by using the HPLC analysis (Reto,
Figueira, Filipe, & Almeida, 2007).
Statistical analysis
The ANOVA and following mean multiple comparisons
using the Tukey HSD intervals (a ¼ 0.05) of all data revealed
a wide and high variability between each type of tea and
within each group of tea (Data not shown). This confirms
what was stated above regarding observed variability of
analyzed parameters among tea samples. Manning and
Roberts (2003) and Pongsuwan et al. (2007) also found
similar statistical variability when analyzed different com-
mercial green tea products.
The PCA was performed to clarify and better analyze
obtained results. PCA reduces the data dimensionality and
allows their visualization retaining as much as possible the
variability present in the original data (Martens & Martens,
2001).
The first PCA model retained two principal components
(t1 and t2) which explained 83.8% of the total variability of
dataset. As shown in Figure 2, t1 (57.6% of explained
variance) arranges samples (left to right) according to their
high total phenolics, EGCG, ECG, and GA concentrations;
meanwhile component t2 (26.2% of explained variance)
arranges samples (top to bottom) according to their high
a-amylase inhibitory activities and pH values. These results
reveal that analyzed tea samples showed similar patterns
depending on their manufacture conditions (type of tea) and
CyTA – Journal of Food 65
Figure 2. Score plot for PCA model.
Figura 2. Gráfica de resultados de modelo PCA (Análisis de
componentes principales).
brand. Overall, black and red teas formed two properly
defined groups (Groups A and B, respectively), however, a
wider dispersion was observed within the black tea group (A),
possibly associated to the differences in fermentation condi-
tions, origin, or agronomic factors. The Twinings Darjeeling
black tea (Group G) was the only sample with a different and
a unique pattern compared to the rest of analyzed black teas
and even to the other tea groups. Therefore, this specific
brand of black tea likely corresponds to a new type of tea.
This sample also showed the highest total phenolic content
and had a different HPLC phenolic profile among black teas.
Such observed trend could be difficult to explain and may be
related either to its origin or to a not declared mixture of teas.
However, more studies would be necessary to confirm this.
No defined groups were observed among green and white
teas (C and D groups and E and F groups). Only the Akbar
green (Group F) and the Twinings white teas (Group E)
showed different patterns. This would be associated to their
high total phenolic contents as discussed above. The Dilmah
oolong tea showed a particular pattern close to the green
group (Group H). In this case, the manufacture conditions
(semi-fermentation) determine the oolong tea differentiation.
The supervised pattern recognition technique PLS-DA
was applied to confirm relationships suggested by the PCA.
In case of current data, four groups were designated: class 1
(black teas), class 2 (red teas), class 3 (green teas), and class 4
(white teas). Since the Twinings Darjeeling black tea showed a
different pattern, data from this sample were excluded in the
analysis.
The PLS-DA model (Figure 3) fitted four PLS-compo-
nents which explained 96.6% of X variance, while the
explained variance for Y vector reached over 65.2%. Further,
the cumulative overall cross-validated Q2 was 62.5%. The
PLS-DA model (t1 versus t2) confirmed the separation of
black and red teas classes, while green and white classes were
not well discriminated. However, the PLS-DA model
properly classified the 100% of black teas, 88.89% of green
teas, 69.44% of red teas, and only 26.3% of white teas
(85.62% weighted average by sample size). This would be
 66 A.M. Quesille-Villalobos et al.
Downloaded by [University of Dayton] at 21:25 28 December 2014
Figure 3. Score plot for PLS-DA model (t1 vs. t2).
Figura 3. Gráfica de resultados de modelo PLS-DA (Mı́nimos Cuadrados Parciales – Análisis Discriminante) (t1 v/s t2).
related to the overlapping pattern observed in white and
green tea classes.
Conclusions
The current study revealed a high variability among analyzed
tea products commercialized in Chile. Overall, high total
phenolic contents and DPPH radical-linked antioxidant
capacities were related to white and green teas. Black teas
showed a significant in vitro a-amylase inhibitory activity
indicating potential anti-hyperglycemic properties. Major
catechins found in white and green teas were EGCG and
ECG, whereas GA was mainly detected in fully fermented
teas. Independently of the type of tea, CA was found in all tea
samples. According to the PCA and PLS-DA multivariate
analysis of all data, black and red teas formed two defined
groups whereas green and white teas did not exhibited a clear
separation. Such patterns were significantly related to the
total phenolic contents, HPLC phenolic profiles, and a-
amylase inhibitory activities, which in turn would indicate
both influence of manufacture conditions (type of tea) and
brand. This is the first time that tea products commercialized
in Chile are evaluated considering their phenolic profiles, CA
contents, and some health-relevant in vitro properties.
Acknowledgement
We thank the Direction of Research and Innovation from the
Pontificia Universidad Catolica de Valparaiso for the grant
No. 037.225/2009 to L.G.R.
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