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��Food Research International 53 (2013) 891 899 Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres Phenolic compounds in Cistus incanus herbal infusions Antioxidant capacity and thermal stability during the brewing process Peer Riehle, Maren Vollmer, Sascha Rohn N 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 Received in revised form 7 September 2012 with an associated strong antioxidant activity. For the customers it is of interest, if the advertised phenolic Accepted 14 September 2012 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: Therefore, it is important to consider processing parameters such as brewing water, brewing temperature, Cistus incanus and brewing duration for the preparation of C. incanus herbal infusions. The aims of this study were to Phenolic compounds analyze the phenolic compounds of C. incanus herbal infusions, to estimate the antioxidant capacity of the in- Antioxidant capacity 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 Spitzm�ller, Hannig, & Al-Ahmad, 2009) and reducing the initial bacte- eight genera and 175 species. One of the characteristic genera is Cistus, rial adhesion (Hannig, Spitzm�ller, 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 (Barraj�n-Catal�n 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, Hern�ndez, Alegre, Van Breusegem, and Munn�-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 oxidative-stress associated degenerative diseases, such as cancer, N Corresponding author. Tel.: +49 40 42838 7979; fax: +49 40 42838 4342. E-mail address: rohn@chemie.uni-hamburg.de (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.2012.09.020 892 P. Riehle et al. / Food Research International 53 (2013) 891 899 Morand, R�m�sy, & Jim�nez, 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 (e"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 (D�ren, 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. Barraj�n-Catal�n 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 2.3. Sample preparation for example were described by Crozier, Lean, McDonald, and Black (1997), who observed lower flavonoid contents after boiling vegeta- 0.675 g of C. incanus material was brewed in 45 mL water. Boiling bles. Furthermore, Rohn, Buchner, Driemel, Rauser, and Kroh (2007) duration was varied between 5 min and 1 h and temperature be- observed degradation products of the flavonol quercetin after boiling tween 70 �C and 95 �C. Three different types of water: water of onions at 100 �C in an aqueous medium. Altogether, thermal effects Milli-Q-quality (pH 7.0, total water hardness of 0.0 mM), tap water on phenolic compounds in C. incanus infusions seem to be highly (pH 7.2, total water hardness of 1.0 mM) and mineral water probable. (pH 7.6, total water hardness of 3.2 mM), were used. Total water But not only thermally induced effects are able to lead to a degra- hardness is expressed as total concentration (mM) of calcium and dation of phenolic compounds in tea or herbal infusions. Even other magnesium salts. The pH-values were determined using a pH-meter factors have to be considered. For example, through complexing reac- and the degree of total hardness was determined by the use of tions between phenolic compounds and caffeine, a precipitation with quick test sticks. After centrifugation for 10 min at 3220�g, an ali- corresponding lower phenolic contents in hot beverages occur quot of 35 mL of the extract was taken from the supernatant. Lyoph- (D'Amelio, Fontanive, Uggeri, Suggi-Liverani, & Navarini, 2009; Rob- ilization of the extract and dissolving the solids obtained in 2 mL erts, 1963). Large particle sizes and high tea concentrations promote water followed. The next step in sample preparation included a SPE this effect, the so called tea creaming in black tea (J�bstl et al., 2005). (Solid Phase Extraction) according to the method of Breitfellner, The aims of this work were the determination of the phenolic com- Solar, and Sontag (2002) using polyamide 6 as stationary phase and pound profile, the estimation of the contribution of the single phenolic two elutions, one with methanol and one with ammonia containing substances to the antioxidant capacity, as well as the identification of methanol, for purification and fractionation. After drying under a the influence of brewing parameters on the stability of the phenolic stream of nitrogen overnight, redissolving in 2 mL methanol 70% compounds during the preparation of C. incanus infusions. The phenolic and syringe filtration (0.45 �m nylon membrane), the extracts were substances of C. incanus tea, brewed under different conditions, were analyzed with LC DAD/ESI MS/MS and LC onlineTEAC. 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- 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 (e"90%), gallocatechin (e"98%), epigallocatechin a Luna� 5 �m C18 100 � (150� 3.00 mm) column equipped with a (e" 90%), myricitrin (e"99%), quercitrin hydrate (e" 78%), rosmarinic C18 security guard (4� 3.00 mm), both from Phenomenex Inc. acid (e"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 (2,22 -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; e" 98%) and potassium peroxo- A binary gradient system with eluent (A) 0.1% formic acid in water, disulfate (e"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 (e"98%), was used for methanolic SPE eluates: 5% B isocratic (0 2 min), 5 10% formic acid (e"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 P. Riehle et al. / Food Research International 53 (2013) 891 899 893 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 MSn weight. The identification of the phenolic compounds in C. incanus tea in- fusions was carried out on a LC DAD/ESI MSn system consisting of an 2.5. Statistical analysis UHPLC Ultimate 3000 RS with Chromeleon 6.8 software (Dionex GmbH, Idstein, Germany), and an ESI MSn 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 �mphenyl hexyl 100 � (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 �m C18 100 � (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 (4�-8)-gallocatechin or the regio-isomer gallocatechin-(4�-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-(4�-8)-catechin isocratic (65 70 min), 95 10% B (70 75 min), and 10% B isocratic or catechin-(4�-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 �MABTS" + 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 �MABTS" + 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, 894 P. Riehle et al. / Food Research International 53 (2013) 891 899 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-(4�-8)-gallocatechin or Gallocatechin-(4�-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-(4�-8)-catechin or catechin-(4�-8)-gallocatechin1 594 593 575 467 425 407 289 5.9 MeOH 5 Gallocatechin-(4�-8)-catechin or catechin-(4�-8)-gallocatechin1 594 593 575 467 425 407 289 6.2 MeOH 1 6 Procyanidin B1 or procyanidin B3 578 577 559 451 425 407 289 9.0 MeOH 7 Epigallocatechin 306 305 287 261 221 219 179 9.4 MeOH 1 8 Procyanidin B1 or procyanidin B3 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 63 -O-(4-hydroxycinnamoyl)-astragalin2/3 594 593 447 307 285 257 40.6 MeOH 24 63 -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 �m C18 100 � (150�3.00 mm); MeOH: methanolic SPE eluate; MeOH/NH3: ammonia containing methanolic SPE eluate; su- 1 2 perscript numbers 1 to 6 refer to structures that have been described in the literature previously: according to Petereit et al. (1991); according to Saracini, Tattini, Traversi, 3 4 5 Vincieri, and Pinelli (2005); according to Exarchou, Fiamegos, Beek van, Nanos, and Vervoort (2006); according to Barraj�n-Catal�n et al. (2011); according to Fischer, Carle, 6 7 and Kammerer (2011), according to Fern�ndez-Arroyo, Barraj�n-Catal�n, Micol, Segura-Carretero, and Fern�ndez-Guti�rrez (2009); only 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 (Touri�o 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 �mol 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. P. Riehle et al. / Food Research International 53 (2013) 891 899 895 896 P. Riehle et al. / Food Research International 53 (2013) 891 899 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 �g/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 63 -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 P. Riehle et al. / Food Research International 53 (2013) 891 899 897 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 (J�bstl 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 J�bstl 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 (J�bstl 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 (J�bstl 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 898 P. Riehle et al. / Food Research International 53 (2013) 891 899 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 [�mol 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-(4�-8)-gallocatechin or Gallocatechin-(4�-6)-gallocatechin1 14�3.1 0.8�0.16 MeOH 3 Gallocatechin 311 �14 19�0.2 MeOH 4 Gallocatechin-(4�-8)-catechin or catechin-(4�-8)-gallocatechin1 26�2.5 1.6�0.08 MeOH 5 Gallocatechin-(4�-8)-catechin or catechin-(4�-8)-gallocatechin1 62�7.4 4�0.30 MeOH 1 6 Procyanidin B1 or procyanidin B3 4�0.5 0.3�0.03 MeOH 7 Epigallocatechin 15�1.2 0.9�0.04 MeOH 1 8 Procyanidin B1 or procyanidin B3 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 63 -O-(4-hydroxycinnamoyl)-astragalin2/3 0 0.0 MeOH 24 63 -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; 2 superscript numbers 1 to 6 refer to structures that have been described in the literature previously:1according to Petereit et al. (1991); according to Saracini et al. (2005); 3 4 5 6 7 according to Exarchou et al. (2006); according to Barraj�n-Catal�n et al. (2011); according to Fischer et al. (2011); according to Fern�ndez-Arroyo et al. (2009); only detected in C. incanus herbal infusion sample 2. quercetin and rutin when cooking these substances in an aqueous References model system, has to be considered also for the brewing process Attaguile, G., Russo, A., Campisi, A., Savoca, F., Acquaviva, R., Ragusa, N., et al. (2000). of herbal tea infusions. Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L.. Cell Biology and Toxicology, 16, 83 90. Barraj�n-Catal�n, E., Fern�ndez-Arroyo, S., Rold�n, C., Guill�n, E., Saura, D., Segura- Carretero, A., et al. (2011). A systematic study of the polyphenolic composition of 4. Conclusion aqueous extracts deriving from several Cistus genus species: Evolutionary relation- ship. Phytochemical Analysis, 22, 303 312. This research work showed that C. incanus herbal tea infusions Breitfellner, F., Solar, S., & Sontag, G. (2002). Effect of gamma-irradiation on phenolic include various phenolic compounds from different subclasses. Espe- acids in strawberries. Journal of Food Science, 67, 517 521. Buchner, N., Krumbein, A., Rohn, S., & Kroh, L. W. (2006). Effect of thermal processing cially, the groups of phenolic acids, flavonoids as well as flavan-3-ol on the flavonols rutin and quercetin. Rapid Communications in Mass Spectrometry, derivatives were present. These substances are powerful anti- 20, 3229 3235. oxidants with intense antioxidant capacities, as demonstrated by LC Chao, Y. C., & Chiang, B. H. (1999). Cream formation in semifermented tea. Journal of the Science of Food and Agriculture, 79, 1767 1774. onlineTEAC. Another interesting point in the analysis of C. incanus is Couzinet-Mossion, A., Balayssac, S., Gilard, V., Malet-Martino, M., Potin-Gautier, M., & the existence of single substances in herbal infusions of a specific ori- Behra, P. (2010). Interaction mechanisms between caffeine and polyphenols in gin. This fact may be an important approach for further investigations infusions of Camellia sinensis leaves. Food Chemistry, 119, 173 181. Crozier, A., Jaganath, I. B., & Clifford, M. N. (2009). Dietary phenolics: Chemistry, on authenticity and quality of Cistus herbal infusions. For the prepara- bioavailability and effects on health. Natural Product Reports, 26, 1001 1043. tion of these herbal infusions, the choice of the extracting agent is im- Crozier, A., Lean, M. E. J., McDonald, M. S., & Black, C. (1997). Quantitative analysis of portant. By using water with a high degree of total hardness for the flavonoid content of commercial tomatoes, onions, lettuce, and celery. Journal of Agricultural and Food Chemistry, 45, 590 595. brewing, a lower content of phenolic compounds associated with D'Amelio, N., Fontanive, L., Uggeri, F., Suggi-Liverani, F., & Navarini, L. (2009). NMR significantly decreased antioxidant capacities will be the result. The reinvestigation of the caffeine chlorogenate complex in aqueous solution and in phenolic compound containing precipitates perhaps will rest in the coffee brews. Food Biophysics, 4, 321 330. Droebner, K., Ehrhardt, C., Poetter, A., Ludwig, S., & Planz, O. (2007). CYSTUS052, a tea cup and will not be ingested. However, the bioavailability of such polyphenol-rich plant extract, exerts anti-influenza virus activity in mice. Antiviral precipitates in humans was not yet explored in detail. Nevertheless, Research, 76, 1 10. optimized brewing conditions in the brewing process of C. incanus Ehrhardt, C., Hrincius, E. R., Korte, V., Mazur, I., Droebner, K., Poetter, A., et al. (2007). 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Pomponio, R., Gotti, R., Santagati, N., & Cavrini, V. (2003). Analysis of catechins in extracts Fern�ndez-Arroyo, S., Barraj�n-Catal�n, E., Micol, V., Segura-Carretero, A., & of Cistus species by microemulsion electrokinetic chromatography. Journal of Fern�ndez-Guti�rrez, A. (2009). High-performance liquid chromatography with Chromatography. A, 990, 215 223. diode array detection coupled to electrospray time-of-flight and ion-trap tandem Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure antioxidant activity mass spectrometry to identify phenolic compounds from a Cistus ladanifer aqueous relationships of flavonoids and phenolic acids. Free Radical Biology & Medicine, 20, extract. Phytochemical Analysis, 21, 307 313. 933 956. Fiol, M., Adermann, S., Neugart, S., Rohn, S., M�gge, C., Schreiner, M., et al. (2012). Roberts, E. A. H. (1963). The phenolic substances of manufactured tea. X. The Highly glycosylated and acylated flavonols isolated from kale (Brassica oleracea creaming down of tea liquors. Journal of the Science of Food and Agriculture, 14, var. sabellica) Structure antioxidant activity relationship. Food Research Interna- 700 705. tional, 47, 80 89. Rohn, S., Buchner, N., Driemel, G., Rauser, M., & Kroh, L. W. (2007). Thermal degradation Fischer, A. U., Carle, R., & Kammerer, D. R. (2011). Identification and quantification of of onion quercetin glucosides under roasting conditions. Journal of Agricultural and phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril Food Chemistry, 55, 1568 1573. and differently produced juices by HPLC DAD ESI/MSn. Food Chemistry, 127, Santagati, N. A., Salerno, L., Attaguile, G., Savoca, F., & Ronsisvalle, G. (2008). Simultaneous 807 821. determination of catechins, rutin, and gallic acid in Cistus species extracts by HPLC Halliwell, B., Murcia, M. A., Chirico, S., & Aruoma, O. I. (1995). Free radicals and antiox- with diode array detection. Journal of Chromatographic Science, 46, 150 156. idants in food and in vivo: What they do and how they work. Critical Reviews in Saracini, E., Tattini, M., Traversi, M. L., Vincieri, F. F., & Pinelli, P. (2005). Simultaneous Food Science and Nutrition, 35, 7 20. LC DAD and LC MS determination of ellagitannins, flavonoid glycosides, and Hannig, C., Sorg, J., Spitzm�ller, B., Hannig, M., & Al-Ahmad, A. (2009). Polyphenolic acyl glycosyl flavonoids in Cistus salvifolius L. leaves. Chromatographia, 62, beverages reduce initial bacterial adherence to enamel in situ. Journal of Dentistry, 245 249. 37, 560 566. Scalbert, A., Manach, C., Morand, C., R�m�sy, C., & Jim�nez, L. (2005). Dietary polyphenols Hannig, C., Spitzm�ller, B., Al-Ahmad, A., & Hannig, M. (2008). Effects of Cistus-tea on and the prevention of diseases. 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