FRIN-04262; No of Pages 9

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

in the oral cavity by decreasing the amount of bacteria (Hannig, Sorg,
The plant order Cistaceae includes herbaceous plants and shrubs of Spitzmüller, Hannig, & Al-Ahmad, 2009) and reducing the initial bacte-
eight genera and 175 species. One of the characteristic genera is Cistus, rial adhesion (Hannig, Spitzmüller, Al-Ahmad, & Hannig, 2008). For the
growing preferably on degraded areas in the Mediterranean region customers it is of interest, if the advertised phenolic contents are
(Attaguile et al., 2000). In traditional folk medicine Cistus is used in present and if the requested effects can be obtained through ingestion
anti-inflammatory, antiulcerogenic, wound healing, antimicrobial, of the mentioned products.
cytotoxic and vasodilator remedies (Barrajón-Catalán et al., 2011). To understand the several positive and negative effects of antiox-
The main components of this natural medicine are phenolic com- idants, a definition of these compounds is necessary. According to
pounds of the flavonoid family (Pomponio, Gotti, Santagati, & Cavrini, Hernández, Alegre, Van Breusegem, and Munné-Bosch (2009),
2003). Currently, numerous manufacturers offer Cistus incanus herbal plant-derived antioxidants are molecules, which donate electrons or
infusions (‘Cistus tea’) or dietary supplements consisting of this plant hydrogen atoms. These compounds are able to form less reactive
material or extracts of it. These products are especially promoted antioxidant-derived radicals, which are efficiently quenched by
with regard to a high content and a diverse profile of phenolic other electron or hydrogen sources to prevent cellular damage.
substances together with an associated strong antioxidant activity or Furthermore, plant-derived antioxidants are hypothesized to be
further potential health-beneficial effects. For example, aqueous ex- protective against oxidative stress events. In the human diet, phenolic
tracts of C. incanus showed protective effects against DNA cleavage in compounds primarily flavonoids and phenolic acids, are the main an-
cell culture (Attaguile et al., 2000) or anti-influenza virus activities in tioxidants. The estimated daily total dietary intake is thought to reach
mice (Droebner, Ehrhardt, Poetter, Ludwig, & Planz, 2007; Ehrhardt et from 20 mg to 1 g (Rice-Evans, Miller, & Paganga, 1996). Because of
al., 2007). Furthermore, the use of Cistus tea as a biological antibacterial their antioxidant activity, phenolic compounds may protect human
cells against oxidative damage, leading to a reduced risk of several
⁎ Corresponding author. Tel.: +49 40 42838 7979; fax: +49 40 42838 4342. oxidative-stress associated degenerative diseases, such as cancer,
E-mail address: [email protected] (S. Rohn). 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 of total water hardness) were purchased from Carl Roth GmbH & Co. KG
antioxidants, phenolic compounds are, unlike the vitamins, not yet (Karlsruhe, Germany). Rutin trihydrate (≥97%) and gallic acid (98%)
classified as essential for short-term human well-being. The human were purchased from Acros Organics BVBA (Geel, Belgium) and DMSO
body even possesses efficient eliminating mechanisms for these xe- (dimethylsulfoxide) from Honeywell Holding GmbH (Offenbach,
nobiotic compounds (Crozier, Jaganath, & Clifford, 2009). However, Germany). Quercetin-3-O-glucoside was purchased from Extrasynthese
antioxidant effects of phenolic compounds should be regarded SAS (Genay, France). Ammonia solution (25% p.a.) was purchased from
under a controversial point of view. Especially, the frequently pro- Th. Geyer GmbH & Co. KG. (Renningen, Germany). Methanol and aceto-
moted positive effects of phenolic substances led to an increasing in- nitrile were purchased from VWR International GmbH (Darmstadt, Ger-
gestion of food and food supplements with high total phenolic many). All solvents were of LC grade and water was of Milli-Q-quality.
contents. Although no acute toxic effects have been reported until Polyamide 6 (particle size 0.05–0.16 mm) was purchased from
today, pro-oxidative reactions, associated with an overproduction of Macherey–Nagel GmbH & Co. KG (Düren, Germany).
radicals have to be considered. For example, a reduction of Fe(III) to
Fe(II) results in hydroxyl radicals, the most reactive oxygen radicals 2.2. Plant material
(Halliwell, Murcia, Chirico, & Aruoma, 1995) in the so called Fenton-
reaction (Scalbert et al., 2005). C. incanus infusions and products in- Two commercially available samples of C. incanus herbal infusions
cluding extracts of it are notable examples for such promoted were analyzed in this work. Sample 1 was a C. incanus organic herbal
polyphenol-rich food. Barrajón-Catalán et al. (2011) reported the ex- infusion, with coarse-grained inhomogeneous particles (size of about
istence of monomeric and polymeric flavanols, gallic acid, rutin as 2–15 mm). Leaf particles, numerous large stem parts (up to 15 mm)
well as other flavonol glycosides based on quercetin, kaempferol as well as whole blossoms were recognizable. The plants were
and myricetin in C. incanus. grown in Greece (according to manufacturer's information). Sample
With regard to these various phenolic acids and flavonoids, there 2 was a C. incanus herbal infusion, with homogeneous close-grained
is, however, only limited information about the reactivity and stabil- particles (size of about 2–5 mm). Hackled leaves, blossom parts and
ity of these substances during thermal treatments such as boiling, fry- no visible stem parts were recognizable. The origin of the processed
ing or roasting. That may be a reason why these highly probable Cistus plants was Turkey (according to manufacturer's information).
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 2.3. Sample preparation
(1997), who observed lower flavonoid contents after boiling vegeta-
bles. Furthermore, Rohn, Buchner, Driemel, Rauser, and Kroh (2007) 0.675 g of C. incanus material was brewed in 45 mL water. Boiling
observed degradation products of the flavonol quercetin after boiling duration was varied between 5 min and 1 h and temperature be-
onions at 100 °C in an aqueous medium. Altogether, thermal effects tween 70 °C and 95 °C. Three different types of water: water of
on phenolic compounds in C. incanus infusions seem to be highly Milli-Q-quality (pH 7.0, total water hardness of 0.0 mM), tap water
probable. (pH 7.2, total water hardness of 1.0 mM) and mineral water
But not only thermally induced effects are able to lead to a degra- (pH 7.6, total water hardness of 3.2 mM), were used. Total water
dation of phenolic compounds in tea or herbal infusions. Even other hardness is expressed as total concentration (mM) of calcium and
factors have to be considered. For example, through complexing reac- magnesium salts. The pH-values were determined using a pH-meter
tions between phenolic compounds and caffeine, a precipitation with and the degree of total hardness was determined by the use of
corresponding lower phenolic contents in hot beverages occur quick test sticks. After centrifugation for 10 min at 3220 × g, an ali-
(D'Amelio, Fontanive, Uggeri, Suggi-Liverani, & Navarini, 2009; Rob- quot of 35 mL of the extract was taken from the supernatant. Lyoph-
erts, 1963). Large particle sizes and high tea concentrations promote ilization of the extract and dissolving the solids obtained in 2 mL
this effect, the so called tea creaming in black tea (Jöbstl et al., 2005). water followed. The next step in sample preparation included a SPE
The aims of this work were the determination of the phenolic com- (Solid Phase Extraction) according to the method of Breitfellner,
pound profile, the estimation of the contribution of the single phenolic Solar, and Sontag (2002) using polyamide 6 as stationary phase and
substances to the antioxidant capacity, as well as the identification of two elutions, one with methanol and one with ammonia containing
the influence of brewing parameters on the stability of the phenolic methanol, for purification and fractionation. After drying under a
compounds during the preparation of C. incanus infusions. The phenolic stream of nitrogen overnight, redissolving in 2 mL methanol 70%
substances of C. incanus tea, brewed under different conditions, were and syringe filtration (0.45 μm nylon membrane), the extracts were
analyzed using LC–DAD/ESI–MS/MS. As the well-known photometric analyzed with LC–DAD/ESI–MS/MS and LC–onlineTEAC.
TEAC-assay(s), which are still used in many antioxidant activity studies,
are lacking the important information about which compounds are re- 2.4. Separation, identification and determination of antioxidant capacities
sponsible for the overall antioxidant activity, the LC–onlineTEAC meth-
odology for the evaluation of the antioxidant capacity of single phenolic 2.4.1. LC–DAD
substances was applied. The phenolic compounds of the infusions were analyzed on a LC–
DAD Smartline series system from Knauer GmbH (Berlin, Germany).
2. Experimental The LPG (Low Pressure Gradient) consisted of a Smartline manager
S5050, pump S1000, autosampler S3950 and diode array detector
2.1. Chemicals S2600. The system was controlled by ClarityChrom 3.0 software
(Knauer GmbH, Berlin, Germany). The separation was carried out on
Epicatechin (≥ 90%), gallocatechin (≥ 98%), epigallocatechin a Luna® 5 μm C18 100 Å (150 × 3.00 mm) column equipped with a
(≥ 90%), myricitrin (≥ 99%), quercitrin hydrate (≥ 78%), rosmarinic C18 security guard (4 × 3.00 mm), both from Phenomenex Inc.
acid (≥ 98%), trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2- (Aschaffenburg, Germany), at a temperature of 21 °C, a flow rate of
carboxylic acid; 97%), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline- 0.6 mL/min and detection at 280 nm, 325 nm, 350 nm and 365 nm.
6-sulfonic acid) diammonium salt; ≥ 98%) and potassium peroxo- A binary gradient system with eluent (A) 0.1% formic acid in water,
disulfate (≥ 99%) were purchased from Sigma Aldrich Chemie eluent (B) 0.1% formic acid in acetonitrile and the following gradient
GmbH (Schnelldorf, Germany). Catechin (~CHR), ellagic acid (≥98%), was used for methanolic SPE eluates: 5% B isocratic (0–2 min), 5–10%
formic acid (≥98%) and quick test sticks Aquadur® (for determination B (2–6 min), 10–30% B (6–45 min), 30–95% B (45–55 min), 95% B

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 3

isocratic (55–60 min), 95–5% B (60–65 min), and 5% isocratic peak area at 734 nm of 1 mM internal trolox standard and multiplying
(65–75 min). by 1 mM Trolox. The resulting antioxidant capacity was then calculated
relative to 100 g of C. incanus sample taking into consideration the sample
2.4.2. LC-ESI–MS n weight.
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 2.5. Statistical analysis
UHPLC Ultimate 3000 RS with Chromeleon 6.8 software (Dionex
GmbH, Idstein, Germany), and an ESI–MS n amaZon ETD with soft- Two C. incanus herbal infusions of each sample were brewed and
ware trapControl 7.0, HyStar 3.2 and DataAnalysis 4.0 (all Bruker analyzed twice. The standard deviation was calculated and the aver-
Daltonik GmbH, Bremen, Germany). The UHPLC consisted of binary aged values along with the standard deviations are documented in
pump, autosampler, column compartment and diode array detector. the respective tables.
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– 3. Results and discussion
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 3.1. Phenolic compounds of C. incanus
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 C. incanus infusions contain various phenolic compounds, includ-
a pressure of 55 psi for the nebulizer were set. The automatic MS/ ing phenolic acids, monomeric and dimeric flavan-3-ols, flavonol gly-
MS mode was chosen for the experiments. cosides as well as ellagitannins. Using a LC–DAD/ESI–MS/MS method,
thirty-two phenolic substances could be identified. In Table 1, de-
2.4.3. LC–onlineTEAC tailed MS and MS/MS-data, as well as retention times and substance
This recently developed LC coupling method combines the ad- distribution in the two SPE eluates, are given. Because of this great di-
vantages of a traditional antioxidant activity assay with a chromato- versity of secondary plant metabolites a fractionation of the infusions
graphic separation. With this technique a detection of radical using a SPE method was carried out prior to the chromatographic sepa-
scavenging compounds in mixtures of different substances is possi- ration. The resulting methanolic eluate contained mainly flavonoids as
ble and TEAC values for single compounds can be determined. The shown in Fig. 1, whereas Fig. 2 demonstrates, that the ammonia
LC–onlineTEAC therefore uses the synthetic stable radical ABTS •+ containing methanolic eluate especially included rosmarinic acid,
to detect the radical-scavenging activity of phenolic compounds in ellagic acid and its derivatives, or ellagitannins. Among these
complex matrices (Fiol et al., 2012; Koleva, Niederlander, & van compounds, gallic acid ([M − H]− 169 m/z), epicatechin ([M − H]−
Beek, 2001; Zietz et al., 2010). The LC system was a Smartline series 289 m/z), catechin ([M − H]− 289 m/z), ellagic acid ([M − H]− 301 m/
system from Knauer GmbH (Berlin, Germany). The LPG consisted of a z), epigallocatechin ([M − H]− 305 m/z), gallocatechin ([M − H]−
Smartline manager S5000, pump S1000, autosampler S3950, diode 305 m/z), myricitrin ([M − H]− 463 m/z), hexahydroxydiphenoyl-
array detector S2600 (for detection of the positive peaks, set at glucose ([M − H]− 481 m/z), quercitrin ([M − H]− 447 m/z), and rutin
280 nm, 325 nm, 350 nm and 365 nm), UV detector S2550 (for de- ([M − H]− 609 m/z) were observed in both eluates of samples 1 and
tection of the negative peaks, set at 414 nm and 734 nm), two 2. These results are supported by the results of Santagati, Salerno,
JetStream ovens (one for the column at 21 °C and one for the reaction Attaguile, Savoca, and Ronsisvalle (2008), who determined gallic acid,
capillary at 40 °C), auxiliary pump S100 (for pumping the ABTS•+ solu- gallocatechin, catechin and rutin in C. incanus. Additionally, dimers of
tion) and reaction capillary (5.0 m×0.25 mm). The system was con- flavan-3-ols were identified in the methanolic SPE eluate of C. incanus
trolled by software ClarityChrom 3.0 from Knauer GmbH (Berlin, infusions. For example, an (epi)-gallocatechin dimer with a quasi-
Germany). Separation of methanolic SPE eluates was carried out on a molecular ion with 609 m/z and its characteristic fragments was
Luna® 5 μm phenyl–hexyl 100 Å (250×4.60 mm) column with a flow detected at a retention time of 4.6 min (Table 1). The fragment ion at
rate of 0.7 mL/min and separation of ammonia containing methanolic el- 305 m/z represents [M− H]− of the (epi)-gallocatechin subunit.
uates was carried out on a Luna® 5 μm C18 100 Å (150×3.00 mm) col- According to Petereit, Kolodziej, and Nahrstedt (1991), gallocatechin-
umn with a flow rate of 0.6 mL/min, both columns were equipped with (4α-8)-gallocatechin or the regio-isomer gallocatechin-(4α-6)-
a C18 security guard (4×3.00 mm) (all Phenomenex Inc., Aschaffenburg, gallocatechin are strongly suggested as molecular structure for this
Germany). A binary gradient system with eluent (A) 0.1% formic acid in dimer. At the retention times of 5.9 min and 6.2 min, two dimers,
water, eluent (B) 0.1% formic acid in acetonitrile and the following gradi- consisting of an (epi)-catechin and an (epi)-galloactechin unit, with
ent steps were used for methanolic SPE eluates: 10% B isocratic [M− H]− at 593 m/z and a characteristic fragment at 289 m/z, which
(0–5 min), 10–15% B (5–10 min), 15% B isocratic (10–30 min), 15–20% resulted from the quasi molecular ion of (epi)-catechin subunit were
B (30–40 min), 20–55% (40–60 min), 55–95% B (60–65 min), 95% B observed, too. For this dimeric structure gallocatechin-(4α-8)-catechin
isocratic (65–70 min), 95–10% B (70–75 min), and 10% B isocratic or catechin-(4α-8)-gallocatechin is suggested according to Petereit et
(75–85 min). For ammonia containing methanolic SPE eluates the follow- al. (1991). Furthermore at retention times of 9.0 min and 10.1 min
ing gradient steps were used: 2% B isocratic (0–5 min), 2–15% B two dimers of (epi)-catechin with [M− H]− at 577 m/z as well as the
(5–30 min), 15–30% B (30–50 min), 30–95% B (50–60 min), 95% B quasi-molecular ion of the monomer (epi)-catechin at 289 m/z were
isocratic (60–65 min), 95–2% (65–70 min), and 2% B isocratic recorded. According to Petereit et al. (1991), these flavan-3-ol dimers
(70–80 min). For analyses of methanolic SPE eluates, a 100 μM ABTS•+ in C. incanus are procyanidin B1 or procyanidin B3. All these dimeric
solution was used with a flow rate of 0.7 mL/min and for ammonia structures have been identified only tentatively at the moment, as the
containing methanolic eluates with a flow rate of 0.6 mL/min. For the exact determination of isomers and binding properties of the mono-
ABTS+ solution 54 mg ABTS and 9.4 mg K2S2O8 were weighed into a meric subunits have not been carried out yet (concentrations were
50 mL volumetric flask and filled up with water. The solution was shaken too low for NMR experiments).
thoroughly, and incubated and light protected at room temperature for An exception among the identified substances was rosmarinic
about 20 h. After a dilution with 950 mL water and sonication for acid, which was only present in sample 2 (from Turkey). The compar-
10 min, the 100 μM ABTS•+ working solution was applied for the LC– ative chromatograms of ammonia containing methanolic eluates of
onlineTEAC. As an internal standard, trolox was included in each sample both infusion samples are shown in Fig. 2. Sample 1 with a high
(the final concentration of 1 mM). TEAC was calculated by dividing the content of stem residues yielded lower peak areas of phenolic com-
negative peak area at 734 nm of single phenolic substance by negative pounds than sample 2, which had almost no stem particles. However,

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
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 compared to the standard substance trolox in the LC–onlineTEAC anal-
content, which should also be detectable as high peak areas. These ysis. The negative peak areas demonstrate the antioxidant capacities of
substances are present in the wooden stem parts (manufacturer's in- the separated substances. They reduced ABTS•+ to the colorless ABTS
formation). Furthermore, sample 2 with almost no wooden stem having no absorbance at the wavelength of 734 nm. Compared to the
parts not only had higher levels of phenolic compounds, but also widely used traditional photometric TEAC-assay(s), the LC–onlineTEAC
contained rosmarinic acid. This fact is of interest for quality control methodology does not lack any information concerning the responsibil-
of Cistus teas, where the ratio between wooden stem parts, leaves ity of single compounds to the overall antioxidant activity. In the photo-
and blossoms could be essential parameters for authenticity and metric assay only a total antioxidant activity of a sample can be
product quality. For example in other parts of woody plants, like determined. Table 2 shows the TEAC values for each identified phenolic
pine (Pinus pinaster) bark, antioxidant catechins and oligomeric compound in C. incanus infusion samples applied in this study, as well
procyanidins have been observed (Touriño et al., 2005). But the as their percentage of the total antioxidant activity when considering
flavan-3-ols especially elicit persistent bitterness and astringency. all compounds. The highest TEAC values of 395, 320, 311, 249 and
The catechin monomers had a stronger bitter taste than the polymers, 242 μmol trolox/100 g C. incanus herbal tea infusion were measured
which were more astringent with increasing molecule size (Peleg, for myricitrin, hexahydroxydiphenoyl-glucose, gallocatechin, gallic
Gacon, Schlich, & Noble, 1999). Altogether, the content of woody acid and catechin, respectively (Table 2). These five substances derive
stem parts in C. incanus infusions might be important for consumer from different subclasses of the phenolic compounds. Myricitrin be-
acceptance. longs to the flavonol glycosides, hexahydroxydiphenoyl-glucose is an
ellagitannin, gallocatechin and catechin are flavan-3-ols and gallic acid
3.2. Antioxidant activity of C. incanus is a member of the phenolic acids. Altogether a complex mixture of phe-
nolic substances with specific antioxidant capacities contributed to the
In Fig. 3 LC–onlineTEAC chromatogram of the methanolic eluate of C. total antioxidant activity of C. incanus herbal tea infusions. Additionally,
incanus herbal infusion of sample 1 is shown. The described phenolic it is of importance that even compounds with rather low contents con-
substances are largely responsible for the promoted antioxidant activi- tribute to large parts of total antioxidant activity. For example,
ties of C. incanus infusion, as they showed high antioxidant capacities 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 double bond between positions 2 and 3 and a 4-oxo function in the
in methanolic SPE eluates in this work. According to Santagati et al. C-ring were assumed to be responsible for the high TEAC values. This
(2008) C. incanus only contains 3.1 μg/g of gallocatechin. For this sub- configuration of flavonoid skeleton is important for electron delocaliza-
stance not the concentration but the prerequisite chemical structure is tion across the molecule and for the stability of the phenoxyl radical
the explanation for its comparatively high antioxidant capacity. (Rice-Evans et al., 1996) resulting from the reaction between an antiox-
Rice-Evans et al. (1996) showed that ortho-hydroxyl groups in the phe- idant molecule and ABTS•+. The additional third hydroxyl group in the
nolic B-ring of the flavonoid skeleton are important molecular features B-ring of myricitrin does not enhance the TEAC value any further
for a high antioxidant capacity. In contrast to gallocatechin, compounds (Rice-Evans et al., 1996).
with large peak areas in the UV-chromatograms (280 nm), such as
6″-O-(4-hydroxycinnamoyl)-astragalin (peak no. 23/24) had no antiox- 3.3. Thermal stability of the phenolic compounds of C. incanus
idant capacity, at all. The missing ortho-diphenolic structure in the
B-ring might be responsible for that or the glycosylation of the hydroxyl With regard to the stability of the phenolic compounds, a signifi-
group on position 3 in the C-ring. In general, a glycosylation of flavo- cant decrease was observed, when brewing both commercially avail-
noids reduces their antioxidant capacities (Rice-Evans et al., 1996). able C. incanus infusions with water of Milli-Q-quality, tap water, or
The highest TEAC value in C. incanus herbal tea infusions was exhibited mineral water. The highest levels of phenolic compounds were
by myricitrin (Table 2). Although glycosylated, this rhamnoside of detected when using water of Milli-Q-quality and the lowest peak
myricitin contains the described ortho-diphenolic configuration in the areas of phenolic substances were detected by brewing tea infusions
B-ring and a hydroxyl group in position 3 of C-ring. Additionally, a 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 7

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 process. For example, variations in pH-value from pH 3.4 to pH 6.7
Milli-Q-quality water and mineral water. Fig. 1 shows the three corre- lead from a maximum amount of tea cream to an absence of cream
sponding chromatographic separations for methanolic eluates of formation in black tea (Chao & Chiang, 1999). High total phenolic
sample 1. Because of the almost similar pH-values of the water, contents and the presence of a variety of large oligo- or polymeric
pHs 7.0 to 7.6, other reasons besides alkalinity of the extracting molecules support tea creaming (Couzinet-Mossion et al., 2010). In
agent have to be considered for the changes of the phenolic com- this work, dimeric flavonoid molecules as well as ellagitannins were
pounds of the infusion samples prepared. In this context, it was identified in C. incanus herbal infusions, which may lead to a creaming
shown that with an increasing total hardness and mineral content during the cooling process. Furthermore, calcium content of brewing
of the water (Milli-Q-quality water: total water hardness of water is an important factor for the precipitation of polyphenols in
0.0 mM; tap water: total water hardness of 1.0 mM; mineral water: black tea, too. High calcium contents were associated with a creaming
total water hardness of 3.2 mM), decreasing peak areas of the pheno- in black tea (Jöbstl et al., 2005). Besides this effect, the structure of tea
lic compounds were observable (Fig. 1). Especially, when brewing leaves can also be modified through calcium in brewing water leading
C. incanus herbal tea infusion sample 1 for 1 h at 95 °C in water of to a decreased extraction of phenolic compounds (Couzinet-Mossion
Milli-Q-quality compared to brewing under the same conditions in et al., 2010). According to Jöbstl et al. (2005), a reduction in tea
mineral water, significant decreases in peak areas for different groups creaming, may be achieved by increasing phenolic compound solubil-
of phenolic compounds were detected. The peak area of gallic acid de- ity or lower calcium contents in the brewing water. Both may lead to
creased almost totally (100%). The decrease in peak areas of the increased total phenolic contents followed by higher antioxidant
flavan-3-ol derivatives (epi)-gallocatechin or (epi)-catechin was up capacities in C. incanus herbal tea infusions.
to 100% and a decrease of 84–100% was detected for the dimers of Besides the kind of brewing water, variations in boiling duration
flavan-3-ols (procyanidins B1 and B3). For the flavonol glycosides and temperature reached significant effects in extraction yields, too.
such as myricitrin or quercitrin, an overall decrease in peak areas be- A positive correlation between boiling time, boiling temperature
tween 9 and 95% was measured. In summary, for all 24 substances of and content of phenolic compounds was observed. By modifying the
the methanolic SPE eluate a total decrease in peak area of 68% was ob- boiling time between 5 min and 1 h under constant conditions of
served (Fig. 1). A reason for this observation can be a so called 95 °C and the use of Milli-Q-quality water, an increasing total peak
‘tea-creaming’ effect resulting from the corresponding mineral con- area of phenolic compounds in the methanolic SPE eluate of 28%
tents in water. Tea cream is a precipitate observed in cooled down was measured. These results strongly suggest a positive correlation
tea (Jöbstl et al., 2005). According to Roberts (1963) such cream is a between extraction time and content of phenolic compounds in
complex of caffeine and theaflavins as well as thearubigins in black C. incanus infusions.
tea (Camellia sinensis). But also decaffeinated black tea is able to With regard to the brewing temperature, a variation from 70 °C
form creams (Penders et al., 1998). Therefore, a formation of tea to 95 °C with a brewing time of 5 min in water of Milli-Q-quality
cream in C. incanus is highly expected, because of its high total pheno- led to an increase of 33% in total peak area of all 24 phenolic com-
lic content with especially high concentrations of flavan-3-ol (deriva- pounds of the methanolic SPE eluate. Furthermore, there is a large
tives). This hypothesis is also supported by the results of Chao and variation in the correlation between temperature and content of
Chiang (1999), who showed that flavan-3-ols which are also present phenolic compounds in C. incanus herbal tea infusions. These results
in high contents in C. incanus, were the main components of tea suggest that the extraction of some phenolic substances is more ef-
cream in semi-fermented teas. Additionally, self-association of tea fective at higher temperatures. For example, peak area of catechin
phenolic compounds such as gallic acid or quercitrin is one consider- increased for 76%. On the other side, there were substances,
able factor for tea creaming (Jöbstl et al., 2005). Correspondingly, e.g. myricitrin, which yielded an increase of only 7% in peak area,
these two phenolic compounds were detected in C. incanus herbal when raising the temperature for the brewing process from 70 °C
tea infusions. Generally, tea cream extent is influenced by pH-value, to 95 °C. A thermal degradation of this substance, similar to the
temperature and water to dry matter ratio during the brewing descriptions of Buchner, Krumbein, Rohn, and Kroh (2006) for

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
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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during the brewing process..., Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.09.020
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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