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 b s t r a c t
a r t i c l e i n f o
Article history:
Received 8 August 2012
Received in revised form 7 September 2012
Accepted 14 September 2012
Keywords:
Cistus incanus
Phenolic compounds
Antioxidant capacity
Thermal stability
LC
–DAD/ESI–MS/MS
LC
–onlineTEAC
Currently numerous manufacturers offer herbal infusions or dietary supplements based on the plant Cistus
incanus. These products are especially promoted as offering a high content of phenolic substances together
with an associated strong antioxidant activity. For the customers it is of interest, if the advertised phenolic
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.
Therefore, it is important to consider processing parameters such as brewing water, brewing temperature,
and brewing duration for the preparation of C. incanus herbal infusions. The aims of this study were to
analyze the phenolic compounds of C. incanus herbal infusions, to estimate the antioxidant capacity of the in-
dividual phenolic substances, as well as to investigate the in
fluence of the brewing process on the phenolic
compound pro
file. By the use of LC–DAD/ESI–MS/MS thirty-two phenolic compounds (e.g. phenolic acids,
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 signi
ficant 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
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
(
). In traditional folk medicine Cistus is used in
anti-in
flammatory, antiulcerogenic, wound healing, antimicrobial,
cytotoxic and vasodilator remedies (
The main components of this natural medicine are phenolic com-
pounds of the
flavonoid family (
Pomponio, Gotti, Santagati, & Cavrini,
). 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 pro
file of phenolic
substances together with an associated strong antioxidant activity or
further potential health-bene
ficial effects. For example, aqueous ex-
tracts of C. incanus showed protective effects against DNA cleavage in
cell culture (
) or anti-in
fluenza virus activities in
mice (
Droebner, Ehrhardt, Poetter, Ludwig, & Planz, 2007; Ehrhardt et
). Furthermore, the use of Cistus tea as a biological antibacterial
mouth rinse contributed to the prevention of bio
film induced diseases
in the oral cavity by decreasing the amount of bacteria (
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 de
finition 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 ef
ficiently 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 (
Food Research International 53 (2013) 891
⁎ Corresponding author. Tel.: +49 40 42838 7979; fax: +49 40 42838 4342.
E-mail address:
(S. Rohn).
0963-9969/$
– see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2012.09.020
Contents lists available at
Food Research International
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d r e s
Morand, Rémésy, & Jiménez, 2005
). Despite these positive effects of
antioxidants, phenolic compounds are, unlike the vitamins, not yet
classi
fied as essential for short-term human well-being. The human
body even possesses ef
ficient 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 (
). 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 pro
file 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
, 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-
). Large particle sizes and high tea concentrations promote
this effect, the so called tea creaming in black tea (
).
The aims of this work were the determination of the phenolic com-
pound pro
file, the estimation of the contribution of the single phenolic
substances to the antioxidant capacity, as well as the identi
fication of
the in
fluence 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
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
using polyamide 6 as stationary phase and
two elutions, one with methanol and one with ammonia containing
methanol, for puri
fication 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, identi
fication 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
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P. Riehle et al. / Food Research International 53 (2013) 891
–899
isocratic (55
–60 min), 95–5% B (60–65 min), and 5% isocratic
(65
–75 min).
2.4.2. LC-ESI
–MS
n
The identi
fication 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. N
2
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 K
2
S
2
O
8
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
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 identi
fied. In
, 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
, whereas
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
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 (
). The fragment ion at
305 m/z represents [M
−H]
−
of the (epi)-gallocatechin subunit.
According to
Petereit, Kolodziej, and Nahrstedt (1991)
, gallocatechin-
(4
α-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-(4
α-8)-catechin
or catechin-(4
α-8)-gallocatechin is suggested according to
. 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
, these
flavan-3-ol dimers
in C. incanus are procyanidin B
1
or procyanidin B
3
. All these dimeric
structures have been identi
fied 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 identi
fied 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
. 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,
893
P. Riehle et al. / Food Research International 53 (2013) 891
–899
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 (
). 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 (
). 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
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.
shows the TEAC values for each identi
fied 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 (
). 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 speci
fic 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
Table 1
Phenolic compounds in Cistus incanus herbal infusions: MS-, MS/MS-data and retention times of SPE eluates.
Peak no.
Compound
MW
[g/mol]
[M
−H]
−
[m/z]
MS/MS fragments
[m/z]
R
t
[min]
SPE eluate
1
Gallic acid
170
169
125
2.7
MeOH
2
Gallocatechin-(4
α-8)-gallocatechin or Gallocatechin-(4α-6)-gallocatechin
1
610
609
591
483
441
423
305
4.6
MeOH
3
Gallocatechin
306
305
287
261
221
219
179
5.1
MeOH
4
Gallocatechin-(4
α-8)-catechin or catechin-(4α-8)-gallocatechin
1
594
593
575
467
425
407
289
5.9
MeOH
5
Gallocatechin-(4
α-8)-catechin or catechin-(4α-8)-gallocatechin
1
594
593
575
467
425
407
289
6.2
MeOH
6
Procyanidin B
1
or procyanidin B
3
1
578
577
559
451
425
407
289
9.0
MeOH
7
Epigallocatechin
306
305
287
261
221
219
179
9.4
MeOH
8
Procyanidin B
1
or procyanidin B
3
1
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-galactoside
2
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-xyloside
2
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-galactoside
2
464
463
445
301
179
23.3
MeOH
18
Quercetin-3-O-glucoside
464
463
301
179
23.6
MeOH
19
Quercetin-O-xyloside
2
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)-astragalin
2/3
594
593
447
307
285
257
40.6
MeOH
24
6
″-O-(4-hydroxycinnamoyl)-astragalin
2/3
594
593
447
307
285
257
42.0
MeOH
25
Methylgallate
184
183
139
3.5
MeOH/NH
3
26
Gentisinic acid-O-glucoside
4
316
315
225
153
109
6.5
MeOH/NH
3
27
Uralenneoside
286
285
153
108
9.7
MeOH/NH
3
28
Hexahydroxydiphenoyl-glucose
4/5
483
482
464
450
406
300
271
23.4
MeOH/NH
3
29
Hexahydroxydiphenoyl-glucose
4/5
482
481
463
449
405
299
270
25.3
MeOH/NH
3
30
Ellagic acid-7-O-xyloside
6
434
433
301
31.9
MeOH/NH
3
31
Ellagic acid
302
301
257
229
185
33.8
MeOH/NH
3
32
Rosmarinic acid
7
360
359
223
197
179
161
135
41.1
MeOH/NH
3
R
t
: retention time on column Phenomenex Luna® 5
μm C18 100 Å (150×3.00 mm); MeOH: methanolic SPE eluate; MeOH/NH
3
: ammonia containing methanolic SPE eluate; su-
perscript numbers 1 to 6 refer to structures that have been described in the literature previously:
1
according to
;
2
according to
;
3
according to
Exarchou, Fiamegos, Beek van, Nanos, and Vervoort (2006)
;
4
according to
Barrajón-Catalán et al. (2011)
;
5
according to
6
according to
Fernández-Arroyo, Barrajón-Catalán, Micol, Segura-Carretero, and Fernández-Gutiérrez (2009)
;
7
only detected in C. incanus herbal infusion
sample 2.
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
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P. Riehle et al. / Food Research International 53 (2013) 891
–899
895
P. Riehle et al. / Food Research International 53 (2013) 891
–899
(280 nm), contributes to 19% of total TEAC of the identi
fied compounds
in methanolic SPE eluates in this work. According to
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.
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 (
).
The highest TEAC value in C. incanus herbal tea infusions was exhibited
by myricitrin (
). Although glycosylated, this rhamnoside of
myricitin contains the described ortho-diphenolic con
figuration 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
con
figuration of flavonoid skeleton is important for electron delocaliza-
tion across the molecule and for the stability of the phenoxyl radical
(
) 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
(
3.3. Thermal stability of the phenolic compounds of C. incanus
With regard to the stability of the phenolic compounds, a signi
fi-
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
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
896
P. Riehle et al. / Food Research International 53 (2013) 891
–899
were between the levels resulting from the experiments with
Milli-Q-quality water and mineral water.
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 (
). 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, signi
ficant 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 B
1
and B
3
). 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 (
). 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 (
). According to
such cream is a
complex of caffeine and thea
flavins as well as thearubigins in black
tea (Camellia sinensis). But also decaffeinated black tea is able to
form creams (
). 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
, 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 (
). Correspondingly,
these two phenolic compounds were detected in C. incanus herbal
tea infusions. Generally, tea cream extent is in
fluenced by pH-value,
temperature and water to dry matter ratio during the brewing
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 (
). High total phenolic
contents and the presence of a variety of large oligo- or polymeric
molecules support tea creaming (
). In
this work, dimeric
flavonoid molecules as well as ellagitannins were
identi
fied 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 (
). Besides this effect, the structure of tea
leaves can also be modi
fied through calcium in brewing water leading
to a decreased extraction of phenolic compounds (
). According to
, 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 signi
ficant 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
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
897
P. Riehle et al. / Food Research International 53 (2013) 891
–899
quercetin and rutin when cooking these substances in an aqueous
model system, has to be considered also for the brewing process
of herbal tea infusions.
4. Conclusion
This research work showed that C. incanus herbal tea infusions
include various phenolic compounds from different subclasses. Espe-
cially, the groups of phenolic acids,
flavonoids as well as flavan-3-ol
derivatives were present. These substances are powerful anti-
oxidants with intense antioxidant capacities, as demonstrated by LC
–
onlineTEAC. Another interesting point in the analysis of C. incanus is
the existence of single substances in herbal infusions of a speci
fic ori-
gin. This fact may be an important approach for further investigations
on authenticity and quality of Cistus herbal infusions. For the prepara-
tion of these herbal infusions, the choice of the extracting agent is im-
portant. By using water with a high degree of total hardness for
brewing, a lower content of phenolic compounds associated with
signi
ficantly decreased antioxidant capacities will be the result. The
phenolic compound containing precipitates perhaps will rest in the
tea cup and will not be ingested. However, the bioavailability of such
precipitates in humans was not yet explored in detail. Nevertheless,
optimized brewing conditions in the brewing process of C. incanus
herbal infusions are very important.
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Table 2
TEAC of phenolic compounds in Cistus incanus herbal tea infusions and their percentage of total antioxidant activity of all phenolic compounds identi
fied in selected SPE eluates.
Peak no.
Compound
TEAC
[
μmol trolox/100 g
C. herbal tea infusion]
Percentage of total TEAC
of all phenolic compounds
identi
fied in SPE eluate
[%]
SPE eluate
1
Gallic acid
249 ± 22
15 ± 0.7
MeOH
2
Gallocatechin-(4
α-8)-gallocatechin or Gallocatechin-(4α-6)-gallocatechin
1
14 ± 3.1
0.8 ± 0.16
MeOH
3
Gallocatechin
311 ± 14
19 ± 0.2
MeOH
4
Gallocatechin-(4
α-8)-catechin or catechin-(4α-8)-gallocatechin
1
26 ± 2.5
1.6 ± 0.08
MeOH
5
Gallocatechin-(4
α-8)-catechin or catechin-(4α-8)-gallocatechin
1
62 ± 7.4
4 ± 0.30
MeOH
6
Procyanidin B
1
or procyanidin B
3
1
4 ± 0.5
0.3 ± 0.03
MeOH
7
Epigallocatechin
15 ± 1.2
0.9 ± 0.04
MeOH
8
Procyanidin B
1
or procyanidin B
3
1
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-galactoside
2
93 ± 3.3
6 ± 0.40
MeOH
13
Myricetin-3-O-glucoside
2
15 ± 1.9
0.9 ± 0.09
MeOH
14
Myricetin-O-xyloside
2
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-galactoside
2
25 ± 1.3
2 ± 0.08
MeOH
18
Quercetin-3-O-glucoside
0
0.0
MeOH
19
Quercetin-O-xyloside
2
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)-astragalin
2/3
0
0.0
MeOH
24
6
″-O-(4-hydroxycinnamoyl)-astragalin
2/3
0
0.0
MeOH
Total TEAC of compounds no. 1 to 24
1626 ± 55
25
Methylgallate
16 ± 3.9
3 ± 0.62
MeOH/NH
3
26
Gentisinic acid-O-glucoside
4
14 ± 0.6
2 ± 0.07
MeOH/NH
3
27
Uralenneoside
75 ± 5.6
12 ± 0.8
MeOH/NH
3
28
Hexahydroxydiphenoyl-glucose
4/5
134 ± 4
21 ± 0.5
MeOH/NH
3
29
Hexahydroxydiphenoyl-glucose
4/5
320 ± 4
50 ± 0.9
MeOH/NH
3
30
Ellagic acid-7-O-xyloside
6
7 ± 1.3
1 ± 0.21
MeOH/NH
3
31
Ellagic acid
40 ± 0.7
6 ± 0.08
MeOH/NH
3
32
Rosmarinic acid
7
36 ± 2.3
6 ± 0.32
MeOH/NH
3
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/NH
3
: 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:
1
according to
2
according to
;
3
according to
;
4
according to
Barrajón-Catalán et al. (2011)
;
5
according to
6
according to
Fernández-Arroyo et al. (2009)
7
only detected
in C. incanus herbal infusion sample 2.
898
P. Riehle et al. / Food Research International 53 (2013) 891
–899
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Document Outline
- Phenolic compounds in Cistus incanus herbal infusions — Antioxidant capacity and thermal stability during the brewing process
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