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Food Research International xxx (2012) xxx–xxx

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Food Research International

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Phenolic compounds in Cistus incanus herbal infusions — Antioxidant capacity and

thermal stability during the brewing process
Peer Riehle, Maren Vollmer, Sascha Rohn ⁎
Institute of Food Chemistry, Hamburg School of Food Science, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany

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

Article history: Currently numerous manufacturers offer herbal infusions or dietary supplements based on the plant Cistus
Received 8 August 2012 incanus. These products are especially promoted as offering a high content of phenolic substances together
Accepted 14 September 2012 with an associated strong antioxidant activity. For the customers it is of interest, if the advertised phenolic
Available online xxxx
contents are valid, plant material is authentic and if the suggested effects can be obtained through ingestion.
As it is known from the literature, phenolic compounds can undergo severe changes resulting from cooking.
Keywords:
Cistus incanus
Therefore, it is important to consider processing parameters such as brewing water, brewing temperature,
Phenolic compounds and brewing duration for the preparation of C. incanus herbal infusions. The aims of this study were to
Antioxidant capacity analyze the phenolic compounds of C. incanus herbal infusions, to estimate the antioxidant capacity of the in-
Thermal stability dividual phenolic substances, as well as to investigate the influence of the brewing process on the phenolic
LC–DAD/ESI–MS/MS compound profile. By the use of LC–DAD/ESI–MS/MS thirty-two phenolic compounds (e.g. phenolic acids,
LC–onlineTEAC flavan-3-ol monomers and -dimers as well as flavonol glycosides) were identified. Additionally, specific
antioxidant capacities were attributed to corresponding substances by using the LC–onlineTEAC (Trolox
Equivalent Antioxidant Capacity) methodology. Moreover, the selection of brewing water, boiling time as
well as boiling temperature had a significant influence on the content of the phenolic compounds in
C. incanus infusions. On the basis of these results, it can be concluded, that an incorrect choice of brewing
process parameters could result in a decreased amount of phenolic substances in the final C. incanus beverages
accompanied with a reduced antioxidant activity.
© 2012 Elsevier Ltd. All rights reserved.

1. Introduction mouth rinse contributed to the prevention of biofilm induced diseases

The plant order Cistaceae includes herbaceous plants and shrubs of
eight genera and 175 species. One of the characteristic genera is Cistus,
growing preferably on degraded areas in the Mediterranean region
(Attaguile et al., 2000). In traditional folk medicine Cistus is used in
anti-inflammatory, antiulcerogenic, wound healing, antimicrobial,
cytotoxic and vasodilator remedies (Barrajón-Catalán et al., 2011).
The main components of this natural medicine are phenolic com-
pounds of the flavonoid family (Pomponio, Gotti, Santagati, & Cavrini,
2003). Currently, numerous manufacturers offer Cistus incanus herbal
infusions (‘Cistus tea’) or dietary supplements consisting of this plant
material or extracts of it. These products are especially promoted
with regard to a high content and a diverse profile of phenolic
substances together with an associated strong antioxidant activity or
further potential health-beneficial effects. For example, aqueous ex-
tracts of C. incanus showed protective effects against DNA cleavage in
cell culture (Attaguile et al., 2000) or anti-influenza virus activities in
mice (Droebner, Ehrhardt, Poetter, Ludwig, & Planz, 2007; Ehrhardt et
al., 2007). Furthermore, the use of Cistus tea as a biological antibacterial
⁎ Corresponding author. Tel.: +49 40 42838 7979; fax: +49 40 42838 4342.
E-mail address: rohn@chemie.uni-hamburg.de (S. Rohn).
in the oral cavity by decreasing the amount of bacteria (Hannig, Sorg,
Spitzmüller, Hannig, & Al-Ahmad, 2009) and reducing the initial bacte-
rial adhesion (Hannig, Spitzmüller, Al-Ahmad, & Hannig, 2008). For the
customers it is of interest, if the advertised phenolic contents are
present and if the requested effects can be obtained through ingestion
of the mentioned products.
To understand the several positive and negative effects of antiox-
idants, a definition of these compounds is necessary. According to
Hernández, Alegre, Van Breusegem, and Munné-Bosch (2009),
plant-derived antioxidants are molecules, which donate electrons or
hydrogen atoms. These compounds are able to form less reactive
antioxidant-derived radicals, which are efficiently quenched by
other electron or hydrogen sources to prevent cellular damage.
Furthermore, plant-derived antioxidants are hypothesized to be
protective against oxidative stress events. In the human diet, phenolic
compounds primarily flavonoids and phenolic acids, are the main an-
tioxidants. The estimated daily total dietary intake is thought to reach
from 20 mg to 1 g (Rice-Evans, Miller, & Paganga, 1996). Because of
their antioxidant activity, phenolic compounds may protect human
cells against oxidative damage, leading to a reduced risk of several
oxidative-stress associated degenerative diseases, such as cancer,
cardiovascular or neurodegenerative diseases (Scalbert, Manach,

0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2012.09.020

Please cite this article as: Riehle, P., et al., Phenolic compounds in Cistus incanus herbal infusions — Antioxidant capacity and thermal stability
during the brewing process..., Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.09.020
2 P. Riehle et al. / Food Research International xxx (2012) xxx–xxx
Morand, Rémésy, & Jiménez, 2005). Despite these positive effects of
antioxidants, phenolic compounds are, unlike the vitamins, not yet
classified as essential for short-term human well-being. The human
body even possesses efficient eliminating mechanisms for these xe-
nobiotic compounds (Crozier, Jaganath, & Clifford, 2009). However,
antioxidant effects of phenolic compounds should be regarded
under a controversial point of view. Especially, the frequently pro-
moted positive effects of phenolic substances led to an increasing in-
gestion of food and food supplements with high total phenolic
contents. Although no acute toxic effects have been reported until
today, pro-oxidative reactions, associated with an overproduction of
radicals have to be considered. For example, a reduction of Fe(III) to
Fe(II) results in hydroxyl radicals, the most reactive oxygen radicals
(Halliwell, Murcia, Chirico, & Aruoma, 1995) in the so called Fenton-
reaction (Scalbert et al., 2005). C. incanus infusions and products in-
cluding extracts of it are notable examples for such promoted
polyphenol-rich food. Barrajón-Catalán et al. (2011) reported the ex-
istence of monomeric and polymeric flavanols, gallic acid, rutin as
well as other flavonol glycosides based on quercetin, kaempferol
and myricetin in C. incanus.
With regard to these various phenolic acids and flavonoids, there
is, however, only limited information about the reactivity and stabil-
ity of these substances during thermal treatments such as boiling, fry-
ing or roasting. That may be a reason why these highly probable
changes in the phenolic profile during brewing of tea are often
neglected and not considered. Some data on the stability of flavonoids
for example were described by Crozier, Lean, McDonald, and Black
(1997), who observed lower flavonoid contents after boiling vegeta-
bles. Furthermore, Rohn, Buchner, Driemel, Rauser, and Kroh (2007)
observed degradation products of the flavonol quercetin after boiling
onions at 100 °C in an aqueous medium. Altogether, thermal effects
on phenolic compounds in C. incanus infusions seem to be highly
probable.
But not only thermally induced effects are able to lead to a degra-
dation of phenolic compounds in tea or herbal infusions. Even other
factors have to be considered. For example, through complexing reac-
tions between phenolic compounds and caffeine, a precipitation with
corresponding lower phenolic contents in hot beverages occur
(D'Amelio, Fontanive, Uggeri, Suggi-Liverani, & Navarini, 2009; Rob-
erts, 1963). Large particle sizes and high tea concentrations promote
this effect, the so called tea creaming in black tea (Jöbstl et al., 2005).
The aims of this work were the determination of the phenolic com-
pound profile, the estimation of the contribution of the single phenolic
substances to the antioxidant capacity, as well as the identification of
the influence of brewing parameters on the stability of the phenolic
compounds during the preparation of C. incanus infusions. The phenolic
substances of C. incanus tea, brewed under different conditions, were
analyzed using LC–DAD/ESI–MS/MS. As the well-known photometric
TEAC-assay(s), which are still used in many antioxidant activity studies,
are lacking the important information about which compounds are re-
sponsible for the overall antioxidant activity, the LC–onlineTEAC meth-
odology for the evaluation of the antioxidant capacity of single phenolic
substances was applied.
2. Experimental
2.1. Chemicals
Epicatechin (≥ 90%), gallocatechin (≥ 98%), epigallocatechin
(≥ 90%), myricitrin (≥ 99%), quercitrin hydrate (≥ 78%), rosmarinic
acid (≥ 98%), trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid; 97%), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-
6-sulfonic acid) diammonium salt; ≥ 98%) and potassium peroxo-
disulfate (≥ 99%) were purchased from Sigma Aldrich Chemie
GmbH (Schnelldorf, Germany). Catechin (~CHR), ellagic acid (≥98%),
formic acid (≥98%) and quick test sticks Aquadur® (for determination
Please cite this article as: Riehle, P., et al., Phenolic compounds in Cistus incanus herbal infusions — Antioxidant capacity and thermal stability
during the brewing process..., Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.09.020
of total water hardness) were purchased from Carl Roth GmbH & Co. KG
(Karlsruhe, Germany). Rutin trihydrate (≥97%) and gallic acid (98%)
were purchased from Acros Organics BVBA (Geel, Belgium) and DMSO
(dimethylsulfoxide) from Honeywell Holding GmbH (Offenbach,
Germany). Quercetin-3-O-glucoside was purchased from Extrasynthese
SAS (Genay, France). Ammonia solution (25% p.a.) was purchased from
Th. Geyer GmbH & Co. KG. (Renningen, Germany). Methanol and aceto-
nitrile were purchased from VWR International GmbH (Darmstadt, Ger-
many). All solvents were of LC grade and water was of Milli-Q-quality.
Polyamide 6 (particle size 0.05–0.16 mm) was purchased from
Macherey–Nagel GmbH & Co. KG (Düren, Germany).
2.2. Plant material
Two commercially available samples of C. incanus herbal infusions
were analyzed in this work. Sample 1 was a C. incanus organic herbal
infusion, with coarse-grained inhomogeneous particles (size of about
2–15 mm). Leaf particles, numerous large stem parts (up to 15 mm)
as well as whole blossoms were recognizable. The plants were
grown in Greece (according to manufacturer's information). Sample
2 was a C. incanus herbal infusion, with homogeneous close-grained
particles (size of about 2–5 mm). Hackled leaves, blossom parts and
no visible stem parts were recognizable. The origin of the processed
Cistus plants was Turkey (according to manufacturer's information).
2.3. Sample preparation
0.675 g of C. incanus material was brewed in 45 mL water. Boiling
duration was varied between 5 min and 1 h and temperature be-
tween 70 °C and 95 °C. Three different types of water: water of
Milli-Q-quality (pH 7.0, total water hardness of 0.0 mM), tap water
(pH 7.2, total water hardness of 1.0 mM) and mineral water
(pH 7.6, total water hardness of 3.2 mM), were used. Total water
hardness is expressed as total concentration (mM) of calcium and
magnesium salts. The pH-values were determined using a pH-meter
and the degree of total hardness was determined by the use of
quick test sticks. After centrifugation for 10 min at 3220 × g, an ali-
quot of 35 mL of the extract was taken from the supernatant. Lyoph-
ilization of the extract and dissolving the solids obtained in 2 mL
water followed. The next step in sample preparation included a SPE
(Solid Phase Extraction) according to the method of Breitfellner,
Solar, and Sontag (2002) using polyamide 6 as stationary phase and
two elutions, one with methanol and one with ammonia containing
methanol, for purification and fractionation. After drying under a
stream of nitrogen overnight, redissolving in 2 mL methanol 70%
and syringe filtration (0.45 μm nylon membrane), the extracts were
analyzed with LC–DAD/ESI–MS/MS and LC–onlineTEAC.
2.4. Separation, identification and determination of antioxidant capacities
2.4.1. LC–DAD
The phenolic compounds of the infusions were analyzed on a LC–
DAD Smartline series system from Knauer GmbH (Berlin, Germany).
The LPG (Low Pressure Gradient) consisted of a Smartline manager
S5050, pump S1000, autosampler S3950 and diode array detector
S2600. The system was controlled by ClarityChrom 3.0 software
(Knauer GmbH, Berlin, Germany). The separation was carried out on
a Luna® 5 μm C18 100 Å (150 × 3.00 mm) column equipped with a
C18 security guard (4 × 3.00 mm), both from Phenomenex Inc.
(Aschaffenburg, Germany), at a temperature of 21 °C, a flow rate of
0.6 mL/min and detection at 280 nm, 325 nm, 350 nm and 365 nm.
A binary gradient system with eluent (A) 0.1% formic acid in water,
eluent (B) 0.1% formic acid in acetonitrile and the following gradient
was used for methanolic SPE eluates: 5% B isocratic (0–2 min), 5–10%
B (2–6 min), 10–30% B (6–45 min), 30–95% B (45–55 min), 95% B
 P. Riehle et al. / Food Research International xxx (2012) xxx–xxx
isocratic (55–60 min), 95–5% B (60–65 min), and 5% isocratic
(65–75 min).
2.4.2. LC-ESI–MS n
The identification of the phenolic compounds in C. incanus tea in-
fusions was carried out on a LC–DAD/ESI–MS n system consisting of an
UHPLC Ultimate 3000 RS with Chromeleon 6.8 software (Dionex
GmbH, Idstein, Germany), and an ESI–MS n amaZon ETD with soft-
ware trapControl 7.0, HyStar 3.2 and DataAnalysis 4.0 (all Bruker
Daltonik GmbH, Bremen, Germany). The UHPLC consisted of binary
pump, autosampler, column compartment and diode array detector.
The LC–DAD method was similar to the LC–DAD method as described
above, with the exception of a reduced flow rate of 0.5 mL/min. ESI–
MS/MS experiments were recorded in negative ion mode and a scan-
ning range between 50 and 1500 m/z. The capillary voltage was set to
4.5 kV and the capillary temperature was at 350 °C. N2 was used as
dry gas and helium as collision gas. A dry gas flow of 10 L/min and
a pressure of 55 psi for the nebulizer were set. The automatic MS/
MS mode was chosen for the experiments.
2.4.3. LC–onlineTEAC
This recently developed LC coupling method combines the ad-
vantages of a traditional antioxidant activity assay with a chromato-
graphic separation. With this technique a detection of radical
scavenging compounds in mixtures of different substances is possi-
ble and TEAC values for single compounds can be determined. The
LC–onlineTEAC therefore uses the synthetic stable radical ABTS •+
to detect the radical-scavenging activity of phenolic compounds in
complex matrices (Fiol et al., 2012; Koleva, Niederlander, & van
Beek, 2001; Zietz et al., 2010). The LC system was a Smartline series
system from Knauer GmbH (Berlin, Germany). The LPG consisted of a
Smartline manager S5000, pump S1000, autosampler S3950, diode
array detector S2600 (for detection of the positive peaks, set at
280 nm, 325 nm, 350 nm and 365 nm), UV detector S2550 (for de-
tection of the negative peaks, set at 414 nm and 734 nm), two
JetStream ovens (one for the column at 21 °C and one for the reaction
capillary at 40 °C), auxiliary pump S100 (for pumping the ABTS•+ solu-
tion) and reaction capillary (5.0 m×0.25 mm). The system was con-
trolled by software ClarityChrom 3.0 from Knauer GmbH (Berlin,
Germany). Separation of methanolic SPE eluates was carried out on a
Luna® 5 μm phenyl–hexyl 100 Å (250×4.60 mm) column with a flow
rate of 0.7 mL/min and separation of ammonia containing methanolic el-
uates was carried out on a Luna® 5 μm C18 100 Å (150×3.00 mm) col-
umn with a flow rate of 0.6 mL/min, both columns were equipped with
a C18 security guard (4×3.00 mm) (all Phenomenex Inc., Aschaffenburg,
Germany). A binary gradient system with eluent (A) 0.1% formic acid in
water, eluent (B) 0.1% formic acid in acetonitrile and the following gradi-
ent steps were used for methanolic SPE eluates: 10% B isocratic
(0–5 min), 10–15% B (5–10 min), 15% B isocratic (10–30 min), 15–20%
B (30–40 min), 20–55% (40–60 min), 55–95% B (60–65 min), 95% B
isocratic (65–70 min), 95–10% B (70–75 min), and 10% B isocratic
(75–85 min). For ammonia containing methanolic SPE eluates the follow-
ing gradient steps were used: 2% B isocratic (0–5 min), 2–15% B
(5–30 min), 15–30% B (30–50 min), 30–95% B (50–60 min), 95% B
isocratic (60–65 min), 95–2% (65–70 min), and 2% B isocratic
(70–80 min). For analyses of methanolic SPE eluates, a 100 μM ABTS•+
solution was used with a flow rate of 0.7 mL/min and for ammonia
containing methanolic eluates with a flow rate of 0.6 mL/min. For the
ABTS+ solution 54 mg ABTS and 9.4 mg K2S2O8 were weighed into a
50 mL volumetric flask and filled up with water. The solution was shaken
thoroughly, and incubated and light protected at room temperature for
about 20 h. After a dilution with 950 mL water and sonication for
10 min, the 100 μM ABTS•+ working solution was applied for the LC–
onlineTEAC. As an internal standard, trolox was included in each sample
(the final concentration of 1 mM). TEAC was calculated by dividing the
negative peak area at 734 nm of single phenolic substance by negative
Please cite this article as: Riehle, P., et al., Phenolic compounds in Cistus incanus herbal infusions — Antioxidant capacity and thermal stability
during the brewing process..., Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.09.020
3
peak area at 734 nm of 1 mM internal trolox standard and multiplying
by 1 mM Trolox. The resulting antioxidant capacity was then calculated
relative to 100 g of C. incanus sample taking into consideration the sample
weight.
2.5. Statistical analysis
Two C. incanus herbal infusions of each sample were brewed and
analyzed twice. The standard deviation was calculated and the aver-
aged values along with the standard deviations are documented in
the respective tables.
3. Results and discussion
3.1. Phenolic compounds of C. incanus
C. incanus infusions contain various phenolic compounds, includ-
ing phenolic acids, monomeric and dimeric flavan-3-ols, flavonol gly-
cosides as well as ellagitannins. Using a LC–DAD/ESI–MS/MS method,
thirty-two phenolic substances could be identified. In Table 1, de-
tailed MS and MS/MS-data, as well as retention times and substance
distribution in the two SPE eluates, are given. Because of this great di-
versity of secondary plant metabolites a fractionation of the infusions
using a SPE method was carried out prior to the chromatographic sepa-
ration. The resulting methanolic eluate contained mainly flavonoids as
shown in Fig. 1, whereas Fig. 2 demonstrates, that the ammonia
containing methanolic eluate especially included rosmarinic acid,
ellagic acid and its derivatives, or ellagitannins. Among these
compounds, gallic acid ([M − H]− 169 m/z), epicatechin ([M − H]−
289 m/z), catechin ([M − H]− 289 m/z), ellagic acid ([M − H]− 301 m/
z), epigallocatechin ([M − H]− 305 m/z), gallocatechin ([M − H]−
305 m/z), myricitrin ([M − H]− 463 m/z), hexahydroxydiphenoyl-
glucose ([M − H]− 481 m/z), quercitrin ([M − H]− 447 m/z), and rutin
([M − H]− 609 m/z) were observed in both eluates of samples 1 and
2. These results are supported by the results of Santagati, Salerno,
Attaguile, Savoca, and Ronsisvalle (2008), who determined gallic acid,
gallocatechin, catechin and rutin in C. incanus. Additionally, dimers of
flavan-3-ols were identified in the methanolic SPE eluate of C. incanus
infusions. For example, an (epi)-gallocatechin dimer with a quasi-
molecular ion with 609 m/z and its characteristic fragments was
detected at a retention time of 4.6 min (Table 1). The fragment ion at
305 m/z represents [M− H]− of the (epi)-gallocatechin subunit.
According to Petereit, Kolodziej, and Nahrstedt (1991), gallocatechin-
(4α-8)-gallocatechin or the regio-isomer gallocatechin-(4α-6)-
gallocatechin are strongly suggested as molecular structure for this
dimer. At the retention times of 5.9 min and 6.2 min, two dimers,
consisting of an (epi)-catechin and an (epi)-galloactechin unit, with
[M− H]− at 593 m/z and a characteristic fragment at 289 m/z, which
resulted from the quasi molecular ion of (epi)-catechin subunit were
observed, too. For this dimeric structure gallocatechin-(4α-8)-catechin
or catechin-(4α-8)-gallocatechin is suggested according to Petereit et
al. (1991). Furthermore at retention times of 9.0 min and 10.1 min
two dimers of (epi)-catechin with [M− H]− at 577 m/z as well as the
quasi-molecular ion of the monomer (epi)-catechin at 289 m/z were
recorded. According to Petereit et al. (1991), these flavan-3-ol dimers
in C. incanus are procyanidin B1 or procyanidin B3. All these dimeric
structures have been identified only tentatively at the moment, as the
exact determination of isomers and binding properties of the mono-
meric subunits have not been carried out yet (concentrations were
too low for NMR experiments).
An exception among the identified substances was rosmarinic
acid, which was only present in sample 2 (from Turkey). The compar-
ative chromatograms of ammonia containing methanolic eluates of
both infusion samples are shown in Fig. 2. Sample 1 with a high
content of stem residues yielded lower peak areas of phenolic com-
pounds than sample 2, which had almost no stem particles. However,
4 P. Riehle et al. / Food Research International xxx (2012) xxx–xxx

Table 1
Phenolic compounds in Cistus incanus herbal infusions: MS-, MS/MS-data and retention times of SPE eluates.

Peak no. Compound MW [M − H]− MS/MS fragments Rt SPE eluate

[g/mol] [m/z] [m/z] [min]

1 Gallic acid 170 169 125 2.7 MeOH

2 Gallocatechin-(4α-8)-gallocatechin or Gallocatechin-(4α-6)-gallocatechin1 610 609 591 483 441 423 305 4.6 MeOH
3 Gallocatechin 306 305 287 261 221 219 179 5.1 MeOH
4 Gallocatechin-(4α-8)-catechin or catechin-(4α-8)-gallocatechin1 594 593 575 467 425 407 289 5.9 MeOH
5 Gallocatechin-(4α-8)-catechin or catechin-(4α-8)-gallocatechin1 594 593 575 467 425 407 289 6.2 MeOH
6 Procyanidin B1 or procyanidin B31 578 577 559 451 425 407 289 9.0 MeOH
7 Epigallocatechin 306 305 287 261 221 219 179 9.4 MeOH
8 Procyanidin B1 or procyanidin B31 578 577 559 451 425 407 289 10.1 MeOH
9 Catechin 290 289 271 245 205 179 11.1 MeOH
10 Epicatechin 290 289 271 245 205 179 14.8 MeOH
11 Myricetin-O-rhamnoside-O-hexoside 626 625 607 479 317 271 179 18.6 MeOH
12 Myricetin-3-O-galactoside2 480 479 461 317 271 179 18.8 MeOH
13 Myricetin-3-O-glucoside 480 479 461 317 271 179 19.3 MeOH
14 Myricetin-O-xyloside2 450 449 431 316 271 179 21.9 MeOH
15 Rutin 610 609 591 343 301 271 179 22.4 MeOH
16 Myricitrin 464 463 445 316 179 22.6 MeOH
17 Quercetin-3-O-galactoside2 464 463 445 301 179 23.3 MeOH
18 Quercetin-3-O-glucoside 464 463 301 179 23.6 MeOH
19 Quercetin-O-xyloside2 434 433 301 179 26.2 MeOH
20 Quercitrin 448 447 429 301 179 27.9 MeOH
21 Myricetin-O-rhamnoside-O-hexoside 626 625 607 479 317 271 179 33.0 MeOH
22 Quercetin-O-rhamnoside-O-hexoside 610 609 463 301 179 36.9 MeOH
23 6″-O-(4-hydroxycinnamoyl)-astragalin2/3 594 593 447 307 285 257 40.6 MeOH
24 6″-O-(4-hydroxycinnamoyl)-astragalin2/3 594 593 447 307 285 257 42.0 MeOH
25 Methylgallate 184 183 139 3.5 MeOH/NH3
26 Gentisinic acid-O-glucoside4 316 315 225 153 109 6.5 MeOH/NH3
27 Uralenneoside 286 285 153 108 9.7 MeOH/NH3
28 Hexahydroxydiphenoyl-glucose4/5 483 482 464 450 406 300 271 23.4 MeOH/NH3
29 Hexahydroxydiphenoyl-glucose4/5 482 481 463 449 405 299 270 25.3 MeOH/NH3
30 Ellagic acid-7-O-xyloside6 434 433 301 31.9 MeOH/NH3
31 Ellagic acid 302 301 257 229 185 33.8 MeOH/NH3
32 Rosmarinic acid7 360 359 223 197 179 161 135 41.1 MeOH/NH3

Rt: retention time on column Phenomenex Luna® 5 μm C18 100 Å (150 × 3.00 mm); MeOH: methanolic SPE eluate; MeOH/NH3: ammonia containing methanolic SPE eluate; su-
perscript numbers 1 to 6 refer to structures that have been described in the literature previously: 1according to Petereit et al. (1991); 2according to Saracini, Tattini, Traversi,
Vincieri, and Pinelli (2005); 3according to Exarchou, Fiamegos, Beek van, Nanos, and Vervoort (2006); 4according to Barrajón-Catalán et al. (2011); 5according to Fischer, Carle,
and Kammerer (2011), 6according to Fernández-Arroyo, Barrajón-Catalán, Micol, Segura-Carretero, and Fernández-Gutiérrez (2009); 7only detected in C. incanus herbal infusion
sample 2.

sample 1 in particular has been promoted as having a high phenolic
content, which should also be detectable as high peak areas. These
substances are present in the wooden stem parts (manufacturer's in-
formation). Furthermore, sample 2 with almost no wooden stem
parts not only had higher levels of phenolic compounds, but also
contained rosmarinic acid. This fact is of interest for quality control
of Cistus teas, where the ratio between wooden stem parts, leaves
and blossoms could be essential parameters for authenticity and
product quality. For example in other parts of woody plants, like
pine (Pinus pinaster) bark, antioxidant catechins and oligomeric
procyanidins have been observed (Touriño et al., 2005). But the
flavan-3-ols especially elicit persistent bitterness and astringency.
The catechin monomers had a stronger bitter taste than the polymers,
which were more astringent with increasing molecule size (Peleg,
Gacon, Schlich, & Noble, 1999). Altogether, the content of woody
stem parts in C. incanus infusions might be important for consumer
acceptance.
3.2. Antioxidant activity of C. incanus
In Fig. 3 LC–onlineTEAC chromatogram of the methanolic eluate of C.
incanus herbal infusion of sample 1 is shown. The described phenolic
substances are largely responsible for the promoted antioxidant activi-
ties of C. incanus infusion, as they showed high antioxidant capacities
compared to the standard substance trolox in the LC–onlineTEAC anal-
ysis. The negative peak areas demonstrate the antioxidant capacities of
the separated substances. They reduced ABTS•+ to the colorless ABTS
having no absorbance at the wavelength of 734 nm. Compared to the
widely used traditional photometric TEAC-assay(s), the LC–onlineTEAC
methodology does not lack any information concerning the responsibil-
ity of single compounds to the overall antioxidant activity. In the photo-
metric assay only a total antioxidant activity of a sample can be
determined. Table 2 shows the TEAC values for each identified phenolic
compound in C. incanus infusion samples applied in this study, as well
as their percentage of the total antioxidant activity when considering
all compounds. The highest TEAC values of 395, 320, 311, 249 and
242 μmol trolox/100 g C. incanus herbal tea infusion were measured
for myricitrin, hexahydroxydiphenoyl-glucose, gallocatechin, gallic
acid and catechin, respectively (Table 2). These five substances derive
from different subclasses of the phenolic compounds. Myricitrin be-
longs to the flavonol glycosides, hexahydroxydiphenoyl-glucose is an
ellagitannin, gallocatechin and catechin are flavan-3-ols and gallic acid
is a member of the phenolic acids. Altogether a complex mixture of phe-
nolic substances with specific antioxidant capacities contributed to the
total antioxidant activity of C. incanus herbal tea infusions. Additionally,
it is of importance that even compounds with rather low contents con-
tribute to large parts of total antioxidant activity. For example,
gallocatechin, only providing a very small peak in UV-chromatogram

Fig. 1. LC–DAD chromatograms (at 280 nm) of methanolic SPE eluates of phenolic substances in Cistus incanus herbal infusion (sample 1), brewed for 1 h with (A) water of
Milli-Q-quality (pH 7.0, total water hardness of 0.0 mM), (B) mains water (pH 7.2, total water hardness of 1.0 mM), (C) and mineral water (pH 7.6, total water hardness of
3.2 mM). For compound no. refer to Table 1.

Please cite this article as: Riehle, P., et al., Phenolic compounds in Cistus incanus herbal infusions — Antioxidant capacity and thermal stability
during the brewing process..., Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.09.020
 P. Riehle et al. / Food Research International xxx (2012) xxx–xxx 5

Please cite this article as: Riehle, P., et al., Phenolic compounds in Cistus incanus herbal infusions — Antioxidant capacity and thermal stability
during the brewing process..., Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.09.020
6 P. Riehle et al. / Food Research International xxx (2012) xxx–xxx

Fig. 2. LC–onlineTEAC chromatograms (positive peaks at 280 nm, negative peaks at 734 nm) of ammonia containing methanolic SPE eluates of Cistus incanus herbal infusions
(A) sample 2 and (B) sample 1, including 1 mM trolox as internal standard. For compound no. refer to Table 1.

(280 nm), contributes to 19% of total TEAC of the identified compounds
in methanolic SPE eluates in this work. According to Santagati et al.
(2008) C. incanus only contains 3.1 μg/g of gallocatechin. For this sub-
stance not the concentration but the prerequisite chemical structure is
the explanation for its comparatively high antioxidant capacity.
Rice-Evans et al. (1996) showed that ortho-hydroxyl groups in the phe-
nolic B-ring of the flavonoid skeleton are important molecular features
for a high antioxidant capacity. In contrast to gallocatechin, compounds
with large peak areas in the UV-chromatograms (280 nm), such as
6″-O-(4-hydroxycinnamoyl)-astragalin (peak no. 23/24) had no antiox-
idant capacity, at all. The missing ortho-diphenolic structure in the
B-ring might be responsible for that or the glycosylation of the hydroxyl
group on position 3 in the C-ring. In general, a glycosylation of flavo-
noids reduces their antioxidant capacities (Rice-Evans et al., 1996).
The highest TEAC value in C. incanus herbal tea infusions was exhibited
by myricitrin (Table 2). Although glycosylated, this rhamnoside of
myricitin contains the described ortho-diphenolic configuration in the
B-ring and a hydroxyl group in position 3 of C-ring. Additionally, a
double bond between positions 2 and 3 and a 4-oxo function in the
C-ring were assumed to be responsible for the high TEAC values. This
configuration of flavonoid skeleton is important for electron delocaliza-
tion across the molecule and for the stability of the phenoxyl radical
(Rice-Evans et al., 1996) resulting from the reaction between an antiox-
idant molecule and ABTS•+. The additional third hydroxyl group in the
B-ring of myricitrin does not enhance the TEAC value any further
(Rice-Evans et al., 1996).
3.3. Thermal stability of the phenolic compounds of C. incanus
With regard to the stability of the phenolic compounds, a signifi-
cant decrease was observed, when brewing both commercially avail-
able C. incanus infusions with water of Milli-Q-quality, tap water, or
mineral water. The highest levels of phenolic compounds were
detected when using water of Milli-Q-quality and the lowest peak
areas of phenolic substances were detected by brewing tea infusions
with mineral water. When using tap water, the levels of peak areas

Please cite this article as: Riehle, P., et al., Phenolic compounds in Cistus incanus herbal infusions — Antioxidant capacity and thermal stability
during the brewing process..., Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.09.020
 P. Riehle et al. / Food Research International xxx (2012) xxx–xxx
Fig. 3. LC–onlineTEAC chromatogram (positive peaks at 280 nm, negative peaks at 734 nm) of methanolic SPE eluate of Cistus incanus herbal infusion (sample 1), including 1 mM
trolox as internal standard. For compound no. refer to Table 1.
were between the levels resulting from the experiments with
Milli-Q-quality water and mineral water. Fig. 1 shows the three corre-
sponding chromatographic separations for methanolic eluates of
sample 1. Because of the almost similar pH-values of the water,
pHs 7.0 to 7.6, other reasons besides alkalinity of the extracting
agent have to be considered for the changes of the phenolic com-
pounds of the infusion samples prepared. In this context, it was
shown that with an increasing total hardness and mineral content
of the water (Milli-Q-quality water: total water hardness of
0.0 mM; tap water: total water hardness of 1.0 mM; mineral water:
total water hardness of 3.2 mM), decreasing peak areas of the pheno-
lic compounds were observable (Fig. 1). Especially, when brewing
C. incanus herbal tea infusion sample 1 for 1 h at 95 °C in water of
Milli-Q-quality compared to brewing under the same conditions in
mineral water, significant decreases in peak areas for different groups
of phenolic compounds were detected. The peak area of gallic acid de-
creased almost totally (100%). The decrease in peak areas of the
flavan-3-ol derivatives (epi)-gallocatechin or (epi)-catechin was up
to 100% and a decrease of 84–100% was detected for the dimers of
flavan-3-ols (procyanidins B1 and B3). For the flavonol glycosides
such as myricitrin or quercitrin, an overall decrease in peak areas be-
tween 9 and 95% was measured. In summary, for all 24 substances of
the methanolic SPE eluate a total decrease in peak area of 68% was ob-
served (Fig. 1). A reason for this observation can be a so called
‘tea-creaming’ effect resulting from the corresponding mineral con-
tents in water. Tea cream is a precipitate observed in cooled down
tea (Jöbstl et al., 2005). According to Roberts (1963) such cream is a
complex of caffeine and theaflavins as well as thearubigins in black
tea (Camellia sinensis). But also decaffeinated black tea is able to
form creams (Penders et al., 1998). Therefore, a formation of tea
cream in C. incanus is highly expected, because of its high total pheno-
lic content with especially high concentrations of flavan-3-ol (deriva-
tives). This hypothesis is also supported by the results of Chao and
Chiang (1999), who showed that flavan-3-ols which are also present
in high contents in C. incanus, were the main components of tea
cream in semi-fermented teas. Additionally, self-association of tea
phenolic compounds such as gallic acid or quercitrin is one consider-
able factor for tea creaming (Jöbstl et al., 2005). Correspondingly,
these two phenolic compounds were detected in C. incanus herbal
tea infusions. Generally, tea cream extent is influenced by pH-value,
temperature and water to dry matter ratio during the brewing
Please cite this article as: Riehle, P., et al., Phenolic compounds in Cistus incanus herbal infusions — Antioxidant capacity and thermal stability
during the brewing process..., Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.09.020
7
process. For example, variations in pH-value from pH 3.4 to pH 6.7
lead from a maximum amount of tea cream to an absence of cream
formation in black tea (Chao & Chiang, 1999). High total phenolic
contents and the presence of a variety of large oligo- or polymeric
molecules support tea creaming (Couzinet-Mossion et al., 2010). In
this work, dimeric flavonoid molecules as well as ellagitannins were
identified in C. incanus herbal infusions, which may lead to a creaming
during the cooling process. Furthermore, calcium content of brewing
water is an important factor for the precipitation of polyphenols in
black tea, too. High calcium contents were associated with a creaming
in black tea (Jöbstl et al., 2005). Besides this effect, the structure of tea
leaves can also be modified through calcium in brewing water leading
to a decreased extraction of phenolic compounds (Couzinet-Mossion
et al., 2010). According to Jöbstl et al. (2005), a reduction in tea
creaming, may be achieved by increasing phenolic compound solubil-
ity or lower calcium contents in the brewing water. Both may lead to
increased total phenolic contents followed by higher antioxidant
capacities in C. incanus herbal tea infusions.
Besides the kind of brewing water, variations in boiling duration
and temperature reached significant effects in extraction yields, too.
A positive correlation between boiling time, boiling temperature
and content of phenolic compounds was observed. By modifying the
boiling time between 5 min and 1 h under constant conditions of
95 °C and the use of Milli-Q-quality water, an increasing total peak
area of phenolic compounds in the methanolic SPE eluate of 28%
was measured. These results strongly suggest a positive correlation
between extraction time and content of phenolic compounds in
C. incanus infusions.
With regard to the brewing temperature, a variation from 70 °C
to 95 °C with a brewing time of 5 min in water of Milli-Q-quality
led to an increase of 33% in total peak area of all 24 phenolic com-
pounds of the methanolic SPE eluate. Furthermore, there is a large
variation in the correlation between temperature and content of
phenolic compounds in C. incanus herbal tea infusions. These results
suggest that the extraction of some phenolic substances is more ef-
fective at higher temperatures. For example, peak area of catechin
increased for 76%. On the other side, there were substances,
e.g. myricitrin, which yielded an increase of only 7% in peak area,
when raising the temperature for the brewing process from 70 °C
to 95 °C. A thermal degradation of this substance, similar to the
descriptions of Buchner, Krumbein, Rohn, and Kroh (2006) for
8 P. Riehle et al. / Food Research International xxx (2012) xxx–xxx

Table 2
TEAC of phenolic compounds in Cistus incanus herbal tea infusions and their percentage of total antioxidant activity of all phenolic compounds identified in selected SPE eluates.

Peak no. Compound TEAC Percentage of total TEAC SPE eluate

[μmol trolox/100 g of all phenolic compounds
C. herbal tea infusion] identified in SPE eluate
[%]

1 Gallic acid 249 ± 22 15 ± 0.7 MeOH

2 Gallocatechin-(4α-8)-gallocatechin or Gallocatechin-(4α-6)-gallocatechin1 14 ± 3.1 0.8 ± 0.16 MeOH
3 Gallocatechin 311 ± 14 19 ± 0.2 MeOH
4 Gallocatechin-(4α-8)-catechin or catechin-(4α-8)-gallocatechin1 26 ± 2.5 1.6 ± 0.08 MeOH
5 Gallocatechin-(4α-8)-catechin or catechin-(4α-8)-gallocatechin1 62 ± 7.4 4 ± 0.30 MeOH
6 Procyanidin B1 or procyanidin B31 4 ± 0.5 0.3 ± 0.03 MeOH
7 Epigallocatechin 15 ± 1.2 0.9 ± 0.04 MeOH
8 Procyanidin B1 or procyanidin B31 0 0.0 MeOH
9 Catechin 242 ± 13 15 ± 1.5 MeOH
10 Epicatechin 8 ± 2.2 0.5 ± 0.12 MeOH
11 Myricetin-O-rhamnoside-O-hexoside 8 ± 1.7 0.5 ± 0.09 MeOH
12 Myricetin-3-O-galactoside2 93 ± 3.3 6 ± 0.40 MeOH
13 Myricetin-3-O-glucoside2 15 ± 1.9 0.9 ± 0.09 MeOH
14 Myricetin-O-xyloside2 57 ± 5.3 4 ± 0.18 MeOH
15 Rutin 9 ± 1.6 0.6 ± 0.09 MeOH
16 Myricitrin 395 ± 24 24 ± 0.5 MeOH
17 Quercetin-3-O-galactoside2 25 ± 1.3 2 ± 0.08 MeOH
18 Quercetin-3-O-glucoside 0 0.0 MeOH
19 Quercetin-O-xyloside2 16 ± 1.0 1 ± 0.08 MeOH
20 Quercitrin 62 ± 2.9 4 ± 0.03 MeOH
21 Myricetin-O-rhamnoside-O-hexoside 10 ± 0.3 0.6 ± 0.04 MeOH
22 Quercetin-O-rhamnoside-O-hexoside 3 ± 0.6 0.2 ± 0.05 MeOH
23 6″-O-(4-hydroxycinnamoyl)-astragalin2/3 0 0.0 MeOH
24 6″-O-(4-hydroxycinnamoyl)-astragalin2/3 0 0.0 MeOH
Total TEAC of compounds no. 1 to 24 1626 ± 55
25 Methylgallate 16 ± 3.9 3 ± 0.62 MeOH/NH3
26 Gentisinic acid-O-glucoside4 14 ± 0.6 2 ± 0.07 MeOH/NH3
27 Uralenneoside 75 ± 5.6 12 ± 0.8 MeOH/NH3
28 Hexahydroxydiphenoyl-glucose4/5 134 ± 4 21 ± 0.5 MeOH/NH3
29 Hexahydroxydiphenoyl-glucose4/5 320 ± 4 50 ± 0.9 MeOH/NH3
30 Ellagic acid-7-O-xyloside6 7 ± 1.3 1 ± 0.21 MeOH/NH3
31 Ellagic acid 40 ± 0.7 6 ± 0.08 MeOH/NH3
32 Rosmarinic acid7 36 ± 2.3 6 ± 0.32 MeOH/NH3
Total TEAC of compound nos. 25 to 32 643 ± 4

TEAC: Trolox Equivalent Antioxidant Capacity; MeOH: methanolic SPE eluate of C. incanus sample 1; MeOH/NH3: ammonia containing methanolic SPE eluate of C. incanus sample 2;
superscript numbers 1 to 6 refer to structures that have been described in the literature previously:1according to Petereit et al. (1991); 2according to Saracini et al. (2005);
3
according to Exarchou et al. (2006); 4according to Barrajón-Catalán et al. (2011); 5according to Fischer et al. (2011); 6according to Fernández-Arroyo et al. (2009); 7only detected
in C. incanus herbal infusion sample 2.

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