Influence of the Leaf Content and Herbal Particle Size on the

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Influence of the Leaf Content and the Herbal Particle Size on the Presence and the Extractability of quantitated Phenolic Compounds in Cistus incanus Herbal Teas Peer Riehle, Nele Rusche, Bodo Saake, and Sascha Rohn J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504119s • Publication Date (Web): 25 Oct 2014 Downloaded from http://pubs.acs.org on October 26, 2014

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Journal of Agricultural and Food Chemistry

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Influence of the Leaf Content and the Herbal Particle Size on the Presence and the

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Extractability of quantitated Phenolic Compounds in Cistus incanus Herbal Teas

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Peer Riehlea, Nele Ruschea, Bodo Saakeb, Sascha Rohna*

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a

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Grindelallee 117, 20146 Hamburg, Germany

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b

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Germany

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*Corresponding author: Sascha Rohn, email: [email protected], phone: +49

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Food Chemistry, HAMBURG SCHOOL OF FOOD SCIENCE, University of Hamburg,

Chemical Wood Technology, University of Hamburg, Leuschnerstr. 91 B, 21031 Hamburg,

40/42838-7979, fax: +49 40/42838-4342

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ABSTRACT

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A variety of Cistus incanus products and thereof a majority of herbal teas are offered by

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manufacturers despite a classification as Novel Food. For a re-evaluation of this legal status, a

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characterization of bioactive ingredients will provide data. These teas consist of varying

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compositions of plant parts and particle sizes. While some include high leaf contents with a

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small particle size, others mainly consist of woody stem parts. For the consumer it is of interest

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which product yields the highest concentrations of bioactive phenolic compounds in the final

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infusions.

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In this study, four commercially available samples were divided into leaves and stems.

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Additionally, one sample was reconstituted in three mixtures of these plant parts. The amount

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of wood was determined by cellulose concentration. The aim was to estimate the influence of

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the plant parts on the concentration of phenolic compounds which were identified by LC-DAD-

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ESI-MS/MS and quantitated by LC-DAD.

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Furthermore, one herbal tea was separated into six fractions with different particle sizes to

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investigate the influence of particle size on the extractability of phenolic compounds. On basis

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of the results, the highest concentrations of bioactive compounds in the infusions were yielded

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when brewing leafy parts with a small particle size.

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KEYWORDS

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Novel Food, wood content, cellulose concentration, bioactivity, LC-ESI-MS/MS, LC-DAD,

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TEAC

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INTRODUCTION

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Cistus incanus is one of 175 species of the Mediterranean plant family Cistaceae.1 Besides

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numerous dietary supplements based on this plant or extracts of it, especially a choice of

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herbal teas is currently merchandized by several manufacturers even despite a persisting

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classification as Novel Food by the European Commission. These herbal products are

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promoted for a diverse profile and high contents of phenolic compounds with high antioxidant

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activities. For the re-evaluation of the Novel Food status of C. incanus a classification of plant

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material including a profiling and characterization of bioactive ingredients seems to be

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mandatory. Traditionally, C. incanus was used in natural and folk medicine2,3 since the 4th

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century BC.4 Extracts of it have been shown to be protective against influenza viruses

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resulting mainly from its phenolic compounds as observed in animal and cell culture studies.5-7

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Furthermore, in a clinical study investigating the treatment of upper respiratory tract infections

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with a C. incanus extract, a reduced average duration and severity of symptoms were

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observed.8 Antibacterial effects, in the form of reduced bacterial colonization of oral hard

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tissues, were observed in a pilot study for a mouth rinse consisting of Cistus tea.9 In a

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previous study, altogether 32 phenolic compounds have been analyzed in C. incanus infusions

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and 30 of these compounds showed comparatively high antioxidant capacities.10 The

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antioxidant activity of a substance can be a suitable chemical marker for its reactivity, stability,

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and its bioactivity. Generally, highly reactive antioxidant phenolic compounds may protect

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human cells against oxidative damage, leading to a reduced risk of several oxidative-stress

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associated degenerative diseases.11 Moreover, protective effects against DNA cleavage have

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been proved in cell cultures, too.1 For tea catechins, several of them also ingredients of C.

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incanus infusions,12 an reduction in ischemic heart disease mortality risk was reported.13 Arts

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et al.13 concluded: “Catechins, whether from tea or other sources, may reduce the risk of

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ischemic heart disease mortality but not of stroke.” However, in that study (total) catechin was 3 ACS Paragon Plus Environment


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defined as the sum of several derivatives (particularly catechin, gallocatechin, epicatechin

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etc.). According to large databases about phenolic compounds, catechin concentrations in tea

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vary between approximately 0.7 mg/100 mL in green tea infusions and 2.45 mg/100 mL in

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black tea infusions14,15,16 Concerning infusions brewed from C. incanus herbal tea comparably

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high catechin concentrations were quantitated herein. Furthermore, an amelioration of obesity-

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induced disorders in obese-diabetic mice,17 antihypertensive as well as vasorelaxant effects in

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rat arteries were shown for the flavonoid tiliroside.18 These preliminary results suggest that C.

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incanus infusions may be applied as an `allrounder´ for improving human health and the

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prevention of degenerative diseases or viral pandemics.

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For the consumers of C. incanus herbal teas, it is of great interest to yield high amounts of

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bioactive phenolic compounds in their infusions. However, the herbal basis for these C.

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incanus products consists of varying compositions of different plant parts. Most of the

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commercially available herbal teas include high amounts of leafy material, but some traditional

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manufacturers especially promote large amounts of woody stem parts in their C. incanus

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herbal teas. As a result, the particle size also varies widely in such products.

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So far, some studies investigated the phenolic compounds in different Cistus species. But in

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these studies only the leaves19,20 or aerial parts of Cistus plants have been analyzed, which

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presumably consisted of an undefined mixture of leaves, few stems as well as some

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blossoms.2,12,21-23 Especially in some regions in the Northern Greece, hot water extracts from

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aerial parts consisting of dominantly woody and less leafy parts of C. incanus have been

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traditionally used as remedies.3 But until today no reliable scientific data seems to be available

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for the distribution of bioactive phenolic compounds in the leaves or the woody stem parts of

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C. incanus.

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With regard to the brewing references recommended (by the manufacturers), hot water

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extractability represents a very critical factor in yielding herbal tea infusions with high 4 ACS Paragon Plus Environment

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concentrations of phenolic compounds. In a previous work, the influence of brewing process

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factors such as the type of water, temperature, and brewing duration on the phenolic

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compounds in C. incanus infusions was demonstrated.10 To augment these results, a potential

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influence of herbal particle size on the extractability of bioactive phenolic compounds in C.

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incanus infusions should be taken into consideration in the current study.

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For investigating the presence of bioactive phenolic compounds in C. incanus herbal tea,

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four commercially available products were manually divided into leaves and woody stems.

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However, there are only a few manufacturers that sell products with a high amount of woody

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stems. But especially these products are highly advertised for their health-preventive

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properties. The resulting infusions were analyzed for the presence and concentration of

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phenolic compounds. Additionally, the separated plant parts were reconstituted into three

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defined mixtures. As chemical-analytical marker, the amount of woody parts of these model

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samples was determined by cellulose concentrations and sugar profiles.

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Furthermore, one C. incanus herbal tea was exemplarily separated by the use of a

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laboratory sieving machine into six fractions of different particle sizes with recognizable leafy

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and woody parts to investigate the extractability and the influence of particle size on the

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phenolic substances in the corresponding infusions. By use of LC-DAD-ESI-MS/MS the

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phenolic compounds were identified and 22 substances were quantitated by LC-DAD. The

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chemical reactivity of these quantitated phenolic compounds was estimated using LC-

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onlineTEAC data published previously for the Trolox Equivalent Antioxidant Capacity of the

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single compounds in C. incanus infusions.10

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The aim of this study was to investigate the influence of the herbal leaf content and particle

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size on the concentration of quantitated bioactive phenolic compounds in C. incanus infusions.

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Besides the importance for the consumers with regard to potential health beneficial effects,



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these data might be of certain importance for a re-evaluation of the persisting Novel Food

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status of C. incanus products in the European Community.

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MATERIAL AND METHODS

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Chemicals. Gallocatechin (≥ 98%), myricitrin (≥ 99%), quercitrin hydrate (≥ 78%), procyanidin

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B1 (≥ 90%), trans-ferulic acid (99%), Copper(II) sulfate pentahydrate (Ph. Eur.) and sodium

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carbonate (99.5%) were purchased from Sigma Aldrich Chemie GmbH (Schnelldorf,

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Germany). Catechin (~CHR), formic acid (≥ 98%), D-(+)-xylose (> 99%) and D-(+)-glucose (≥

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99.5%) were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Tiliroside

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(96%) was purchased from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). Gallic

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acid (98%) was purchased from Acros Organics BVBA (Geel, Belgium). Ellagic acid (≥ 98%)

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and myricetin-3-O-galactoside (≥ 99%) were purchased from Extrasynthese SAS (Genay,

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France). Ammonia solution (25% p.a.) and petroleum ether (40-60 °C) were purchased from

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Th. Geyer GmbH & Co. KG. (Renningen, Germany). Acetone, ethanol, sulfuric acid (> 95%),

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boric acid and bicinchoninic acid were purchased from Fisher Scientific GmbH (Schwerte,

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Germany). Glacial acetic acid, methanol and acetonitrile were purchased from VWR

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International GmbH (Darmstadt, Germany). Potassium tetraborate (99.5%) was purchased

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from ChemPur GmbH (Karlsruhe, Germany). MCI Gel CA08F was purchased from Mitsubishi

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Chemical Corporation (Tokyo, Japan). L-aspartic acid (Ph. Eur.) and amylase was purchased

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from Merck KGaA (Darmstadt, Germany). Amyloglucosidase was purchased from Roche

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Diagnostics GmbH (Mannheim, Germany). Polyamide 6 (particle size 0.05–0.16 mm) was

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purchased from Macherey-Nagel GmbH & Co. KG (Düren, Germany). All solvents were of LC

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grade and water was of Milli-Q-quality.

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Plant material. Four commercially available products of C. incanus herbal teas were used as

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a basis for this study. Leaf particles and woody stem parts as well as some blossoms were

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recognizable in all samples. The average sizes of leaves and stems, the stem diameters, as

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well as the country of origin of the four herbal teas are presented in Table 1. For the

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determination of a possible influence of the leaf and wood contents on the presence of

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phenolic compounds in the infusions, the four herbal samples were manually sorted into

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woody stems and leaves. Furthermore three mixtures out of sample 1 were reconstituted

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exemplarily from the stems (st1; “st” stands for sorted) and leaves (st5), resulting in

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preparations with (st2) 25% (w/w), (st3) 50% (w/w) and (st4) 75% (w/w) leaf content and

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corresponding woody stem contents of 75% (w/w), 50% (w/w) and 25% (w/w). In Figure 1 A

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samples st1, st3 and st5 are shown for the manually sorted herbal teas. In a second series of

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experiments, investigating the extractability of the phenolic compounds with regard to particle

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size, C. incanus herbal tea sample 1 was exemplarily separated by use of a laboratory sieving

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machine Typ 3D from Retsch GmbH (Haan, Germany) into six fractions with the sizes (sample

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sv1; “sv” stands for sieved) 2.5-3.1 mm, (sv2) 2.0-2.5 mm, (sv3) 1.4-2.0 mm, (sv4) 1.0-1.4

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mm, (sv5) 0.5-1.0 mm and (sv6) < 0.5 mm. All fractions contained recognizable parts of leaves

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and stem parts (Figure 1 B).

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Sample preparation. 1.25 g of C. incanus herbal tea was brewed in 45 mL water at a

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temperature of 95 °C for 5 min. The brewing time and temperature were used according to

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manufacturer recommendations for the preparation of the infusions. After centrifugation

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(centrifuge 5810 R, Eppendorf GmbH, Hamburg, Germany) for 10 min at 3220 x g, an aliquot

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of 35 mL was taken of the supernatant. Lyophilization of the aliquot using a lyophilizer (Alpha

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1-4 LSC, Martin Christ GmbH, Osterode, Germany) and dissolving the dry residues in 4 mL of

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water followed. The next step in sample preparation included a SPE (Solid Phase Extraction) 7 ACS Paragon Plus Environment


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according to a method of Breitfellner et al.24 using polyamide 6 as stationary phase and two

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elutions, one with methanol and one with ammonia containing methanol, for purification and

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fractionation. After drying under a stream of nitrogen overnight, redissolving in 4 mL of

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methanol 70% and syringe filtration (0.45 µm nylon membrane), the extracts were ready for

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analyses.10 The extracted herb was air-dried at room temperature for about 72 h. An

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Accelerated Solvent Extraction (ASE) with four solvents of different polarity followed for a fully

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extraction of all soluble substances of herb material. The insoluble cellulose was determined

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out of the fully extracted C. incanus herb resulting of brewing process and ASE. The used

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methods for ASE, determination of wood sugar and cellulose concentrations are described

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below.

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Analysis of the wood content. For analyzing the wood content in the sorted leaf and woody

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stem samples (st1-st5), the cellulose concentrations and sugar profiles of the stems were

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measured as analytical markers. Following the determination of the dry weight, 0.5 g of each

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manually sorted sample was extracted in duplicate by use of an Accelerated Solvent

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Extraction System ASE 200 (Dionex GmbH, Idstein, Germany). Two extraction cycles with 10

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mL solvent were performed for 10 min at 70 °C and 10 MPa for each solvent. The following

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solvents were used gradually: petroleum ether, acetone/water (9:1), ethanol/water (8:2) and

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water. After air-drying at room temperature for about 24 h (‘climatization’) and milling with a

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laboratory disc mill Typ T 1000 from Siebtechnik GmbH (Mühlheim, Germany), the ASE

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residues were subjected to a two stage hydrolysis. For the pre-hydrolysis 200 mg of the milled

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material was mixed with 2 mL of 72% sulfuric acid in a reaction tube and hydrolyzed under

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repeated stirring at 30 °C for 1h on a thermostat. By adding 6 mL water the reaction was

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stopped. Afterwards, the pre-hydrolyzed sample was transferred with 50 mL water in a 100 mL

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volumetric flask. For post-hydrolysis the pretreated sample was hydrolyzed at 120 °C and 0.12 8 ACS Paragon Plus Environment

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MPa for 30 min in an autoclave. After cooling, filling to the calibration mark with water and

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subsequent agitation, filtration over a G4 sintered glass crucible and washing the hydrolysis

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residue acid-free with water followed. After drying at 105 °C, the residue was determined

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gravimetrically. With regard to wood chemistry, this residue represents the acid insoluble lignin

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(Klason lignin). The hydrolysates were analyzed with regard to their glucose and xylose

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contents by borate anion exchange chromatography.25 Additionally, a test on starch was

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carried out for the 100% (w/w) woody stem (st1) and the 100% (w/w) leaf sample (st5) to

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evaluate, if the glucose result from cellulose or from starch hydrolysis. For this purpose, the

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two samples described were fine milled by use of a disc mill, 200 mg were weighed in a 25 mL

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volumetric flask and 20 mL of methanol/water (3:1) were added. After shaking thoroughly by

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hand, the solution was incubated for about 15 h and filtrated over a G4 sintered glass filter.

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The filtration residues were washed with 20 mL of methanol/water (3:1) and climatized for 5 d.

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Afterwards, 25 mg of the residues were incubated with 0.75 mL of enzyme solution (0.05%

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amylase and 0.05% amyloglucosidase in 0.1 M ammoniumacetate buffer pH 4.7) at 45 °C for

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24 h. After filtration of the solutions, the glucose concentration was determined by borate anion

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exchange chromatography25 and corrected for an enzyme blank. All monosaccharide and

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starch concentrations were based on the original raw material under consideration of the

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various extraction losses. The cellulose content of samples was calculated from the glucose

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concentration by subtracting the glucose from starch and considering the weight gain due to

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water addition during hydrolysis (-10%).

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Identification and Quantification of the phenolic compounds. Identification of the phenolic

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compounds was previously described by Riehle et al..10 Following SPE clean-up of the C.

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incanus infusions, the phenolic substances in both eluates, one methanolic and one ammonia-

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methanol eluate, were identified by LC-DAD-ESI-MS/MS. 9 ACS Paragon Plus Environment


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For the quantitation of the phenolic compounds, stock solutions of standard substances were

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prepared at a concentration level of 1 mg/mL. For the calibration curves, seven equidistant

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concentrations of each standard were prepared and the purity of standard substances was

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considered in the calculations. A coefficient of determination with R² = 0.99 resulted for all

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calibrations curves. As there are no standards commercially available for all compounds, some

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were quantitated by the use of structural relatives. Thus, methylgallate was quantitated as

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gallic acid and gallocatechin-catechin as procyanidin B1, myricetin-O-xyloside as myricitrin,

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myricetin-3-O-glucoside as myricetin-3-O-galactoside, glycosides of quercetin as quercitrin,

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hexahydroxdiphenoyl-glucose and ellagic acid-7-O-xyloside as ellagic acid, hydroxyferulic

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acid-O-rhamnosides and hydroxyferuloyl-rhamnose as trans-ferulic acid. The corresponding

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correction factors for the difference in molecular weight were included in the calculation of

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substance concentrations. For the quantitation of gallocatechin-catechin as procyanidin B1, it

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has to be considered that the absorption of the gallocatechin subunit was lower compared to

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the absorption of the catechin subunit in procyanidin B1. Consequently, the absolute results

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for gallocatechin-catechin concentrations are slightly higher. For the measurements, the LC-

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DAD method according to Riehle et al.10 was used. Hydroxybenzoic acids, ellagitannins and

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flavanols were detected at a wavelength of 280 nm, hydroxycinnamic acids at 325 nm and the

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flavonols were measured at 350 nm.

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Statistical analysis. Two C. incanus infusions of each sample were brewed and analyzed

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twice. The standard deviation was calculated and the averaged values along with the standard

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deviations are documented in the corresponding figures. The calibration curves for quantitation

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were also analyzed twice.

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RESULTS AND DISCUSSION 10 ACS Paragon Plus Environment

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Identification of the phenolic compounds. C. incanus infusions include a wide range of

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phenolic compounds of different substance classes. According to Riehle et al.,10 32 phenolic

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compounds, belonging to the subclasses of phenolic acids, flavonoids, and ellagitannins were

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detected. Additionally to the previously identified phenolic compounds, a 4-O-rhamnoside and

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5-O-rhamnoside of hydroxyferulic acid as well as a hydroxyferuloyl-rhamnose have been

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tentatively identified by LC-DAD-ESI-MS/MS in this study. Such O-glycosylated phenolic acids

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were also previously identified by Barrajón-Catalán et al.2 for Cistus albidus, one further genus

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out of the plant family Cistaceae. From substances identified, the 22 dominant phenolic

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compounds in the C. incanus infusions have been quantitated with LC-DAD. In Table 2 an

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overview of these compounds, their retention times, MS- and MS/MS-data as well as their

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distribution in the two SPE eluates are shown.

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Leaf and wood contents of the manually sorted samples. For the investigation of the

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influence of herbal leaf and wood content on the composition of phenolic compounds in C.

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incanus infusions, four herbal tea samples were manually sorted into woody stems and leaves.

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Besides samples st1 with 100% (w/w) and st5 with 0% (w/w) woody stem parts, C. incanus

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herbal tea sample 1 was exemplarily reconstituted into three defined mixtures of stems and

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leaves on basis of their dry weight: (st2) 75% (w/w), (st3) 50% (w/w) and (st4) 25% (w/w)

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woody stem parts. In order to monitor the biochemical, ‘real’ wood contents of these

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exemplarily sorted C. incanus herbal teas, the results from acid hydrolysis and carbohydrate

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analysis were considered (Table 3). With regard to wood chemistry, the residue from a sulfuric

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acid hydrolysis represents the acid insoluble lignin content of the sample (Klason lignin).

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However, for the various C. incanus samples, the hydrolysis residue was always in a similar

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order of magnitude and did not correlate with the ratio of stem and leaf material. This indicates

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that other components, e.g. protein from the leaves, are as well incorporated into the residue. 11 ACS Paragon Plus Environment


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Accordingly, this value is not suitable in order to monitor the proportion of leaf and stem

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material. Alternatively, the polysaccharide components of the material have been considered.

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The sum of glucose and xylose combines the main components, representing the cellulose

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and hemicelluloses content of the samples. In addition, the cellulose content was calculated.

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In Table 3, the corresponding values of the five samples (st1-st5) prepared from the leaves

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and stems of sample 1, manually separated and then mixed according to fixed ratios, as well

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as the concentrations of glucose resulting of starch of the 100% (w/w) woody stem (st1) and

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100% (w/w) leaf sample (st5) are presented. Strongly linear positive correlating concentrations

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of glucose and xylose with an increasing amount of woody plant parts (R² = 0.99) were

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revealed. The low glucose values of 1% in the starch test indicated that the glucose, which

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was used for the calculation of cellulose, did not significantly result from starch hydrolysis. On

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basis of the starch test results, a correction factor of 1% was included in the calculation of the

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absolute cellulose concentration. Altogether, the cellulose contents of manually sorted C.

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incanus herbal teas can be used as a valid analytical marker for wood contents.

271 272

Presence of phenolic compounds depending on the plant parts of the herbal tea

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mixture. The presence of the 22 quantitated phenolic compounds in the resulting infusions

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brewed from the four, into woody stems and leaves sorted, herbal tea samples is shown in

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Table 4. In all four samples higher concentrations of quantitated phenolic compounds were

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present in the infusions brewed from the sorted leaves compared to the stems. Furthermore,

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several of the 22 quantitated compounds were even not detectable in the infusions brewed

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from stems. Especially in sample 1, which included the longest stems with the largest

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diameters of the four C. incanus herbal tea samples analyzed, only gallic acid, catechin, and

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methylgallate were detected in infusions brewed from stems. A remarkably exception was the

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catechin concentration. In the four infusions brewed from C. incanus stems, comparatively 12 ACS Paragon Plus Environment

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high concentrations of this phenolic compound from 0.3 mg/100 mL infusion to 1.1 mg/100 mL

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infusion were quantitated. In sample 3, the concentration in the infusion brewed from stems

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(0.9 mg/100 mL) was even higher compared to the infusion brewed from C. incanus leaves

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(0.6 mg/100 mL). Altogether there were differences in the concentrations of the quantitated

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phenolic compounds between the infusions brewed from four C. incanus herbal teas. These

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differences maybe were a result from different plant origins, ecophysiological factors,

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cultivation conditions, or harvest time. For example the plant materials of samples 1 and 2

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were harvested in Greece, sample 4 was from Turkey, and the origin of sample 3 was not

290

available. Discussing the varying presence of phenolic compounds in C. incanus plant parts,

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the plant physiological properties and harvest time of these Mediterranean shrubs should be

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considered, too. These plants have been classified as malakophyllous xerophytes4,26 and are

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drought resistant with seasonal dimorphism of the leaves.27 The plants of this genus develop

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small leaves with numerous trichomes during dry summer periods for minimization of

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transpiration and leaves with only few trichomes in winter.27 According to Christodoulakis et

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al.28, the prominent feature of C. incanus summer leaves is an intense accumulation of

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phenolic secondary metabolites, whereas in less climatically stressed winter leaves, a lower

298

accumulation can be observed. Similar effects have been already shown for environmental

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stress factors such as drought, which significantly increased concentrations of bioactive

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phenolic compounds in Cistus clusii leaves, for example.29 Therefore, investigating the

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influence of seasonal leaf polymorphism on the presence of phenolic compounds in C. incanus

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can be a scientific basis for further research. Altogether, a tentative indication of the

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ecophysiological variation in the concentrations of phenolic compounds in different C. incanus

304

herbal products and plant parts is represented by the results of the four traditional herbal tea

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samples of this work.



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Subsequently, the concentrations of the 22 quantitated phenolic compounds in the infusions

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brewed from the mixtures (st1-st5) of C. incanus sample 1 are shown along six chosen

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phenolic substance classes. In Figure 2, the influence of the leaf content on the concentration

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of phenolic acids (A), flavan-3-ols and their dimers (B), myricetin glycosides (C), quercetin

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glycosides (D), tiliroside isomers (E), and ellagitannins (F) is shown for C. incanus woody

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stems (st1) and leaves (st5) as well as for the three reconstituted mixtures (samples st2-st4).

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The strongest correlations in the class of phenolic acids between leaf content and presence of

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substance resulted for gallic acid (R² = 0.96) and methylgallate (R² = 0.95). Furthermore, these

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two compounds were present in infusions brewed from the 100% (w/w) woody stem sample

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(st1), too. Concentrations for both gallic acid derivatives ranged from 0.1 mg/100 mL infusion

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(st1) to 1.1 mg/100 mL infusion (st5) and for methylgallate of 0.1 mg/100 mL infusion (st1) to

317

0.3 mg/100 mL infusion (st5). This result illustrates that up to 11-fold higher concentrations of

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gallic acid can be yielded when brewing C. incanus infusions from a herbal preparation

319

consisting of 100% (w/w) pure leaves. As it was shown in a previous work, gallic acid is one of

320

the most reactive phenolic compounds in C. incanus infusions, as it is responsible for about

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11% of the total TEAC.10 For the other phenolic acids a similar occurrence in woody stem and

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leaf parts was observed, too.

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With regard to the class of flavan-3-ols (catechins) and their oligomers, the strongest

324

correlation between leaf content of C. incanus herbal tea sample 1 and presence of phenolic

325

substance was found for gallocatechin (R² = 0.99). The range for the gallocatechin

326

concentrations ranged from 0.0 mg/100 mL infusion (st1) to 1.7 mg/100 mL infusion (st5). For

327

the antioxidant capacity and bioactivity of C. incanus infusions, the presence of gallocatechin

328

is an important aspect. This substance contributes to 14% of total TEAC.10

329

In contrast to the gallocatechin concentrations, remarkably high catechin concentrations of

330

0.5 mg/100 mL infusion were quantitated in the sample consisting of 100% (w/w) stems (st1). 14 ACS Paragon Plus Environment

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331

These comparatively high concentrations of catechin in woody parts of C. incanus may also be

332

an argument for the traditional application of the aerial parts, including woody stems, for herbal

333

infusions.3,30 The presence of catechin and further flavan-3-ol derivatives in stems or bark is

334

known for many other plants of different plant families and orders.31-33 Besides the occurrence

335

of catechin in woody plant parts, higher concentrations were reported for leaves.34,35 The

336

presence of catechin in leaves and woody stems of C. incanus also explains the quite low

337

correlation of R² = 0.55 for the influence between the concentrations of this substance and leaf

338

content of reconstituted samples. However, the presence of notable amounts of catechin in

339

infusions brewed from woody stem parts of C. incanus is also important for antioxidant

340

capacity. Catechin was responsible for 11% of total TEAC.10

341

Furthermore, for sensory properties of the infusion, the content of phenolic compounds can

342

be important, too. According to Peleg et al.36, catechin monomers are responsible as they

343

have a strong bitter taste and elicit a slight astringency. However, Scharbert et al.37 described

344

the catechin threshold concentration for astringency being approx. 11.9 mg/100 mL aqueous

345

solution. Bitterness and astringency in C. incanus infusions therefore should only be expected

346

at levels of 10-fold higher catechin concentrations. Furthermore, black and green tea provide

347

astringency and the content of catechin is almost similar in C. incanus infusions as given in

348

polyphenol databases14,15,16 and shown herein. Therefore, the combination and synergisms of

349

different phenolic compounds have been taken into account. Again, Scharbert et al.37 showed

350

for some flavonol glycosides really low sensory threshold levels.

351

For the flavor of herbal infusions it should be also considered that an infusion brewed from

352

100% (w/w) C. incanus leaves exhibits a persistent `green odor´. This odor arises from volatile

353

compounds based on C6-aldehydes and C6-alcohols.38,39

354

With regard to the presence of flavonols in the five reconstituted herbal tea mixtures, the

355

highest concentrations as well as the strongest correlations between leaf contents and 15 ACS Paragon Plus Environment


Page 16 of 34

356

amounts of phenolic compounds in C. incanus infusions were observed for myricitrin and

357

quercitrin. Additionally, both only can be detected in samples that contained pure leaves. For

358

myricitrin, the concentrations reached from 0.0 mg/100 mL infusion (st1) to 1.1 mg/100 mL

359

infusion (st5) and for quercitrin from 0.0 mg/100 mL infusion (st1) to 1.6 mg/100 mL infusion

360

(st5), respectively. The coefficient of determination for the leaf content of sample and

361

concentrations of myricitrin as well as for quercitrin was R² = 0.99. Moreover, high

362

concentrations of myricitrin in C. incanus infusions seem to be an important marker of

363

bioactivity, as it is highly responsible for the infusion’s total antioxidant capacity. This

364

substance contributes to 17% of total TEAC.10

365

A similar distribution in herbal plant material was observed for the isomers of tiliroside. Both

366

isomers were present in samples that only contained leaves (st5), but not in pure woody stems

367

(st1). For the dominant isomer trans-tiliroside, concentrations ranged between 0.0 mg/100 mL

368

infusion (st1) and 0.9 mg/100 mL infusion (st5). The correlation coefficient of R² = 0.99 again

369

suggests a very good linearity between amount of substance and leaf content in a sample.

370

With the exception of catechin an exclusive occurrence of all quantitated flavonoids in C.

371

incanus infusions brewed from leafy parts was observed in this work for sample 1. In woody

372

stem parts these substance class was not present.

373

For the presence of the class of ellagitannins, a similar trend but slightly weaker linear

374

correlation was observed in samples st1 to st5. For the hexahydroxydiphenoyl-glucose

375

isomers concentrations of 0.0 mg/100 mL infusion (st1) to 0.5 mg/100 mL infusion (st5)

376

together with a correlation of R² = 0.76 were detected for the first isomer at retention time of

377

27.1 min. Concentrations of 0.0 mg/100 mL infusion (st1) up to 0.9 mg/100 mL infusion (st5)

378

together with R² = 0.90 were analyzed for the second isomer at retention time of 28.7 min.

379

Both isomers are also important antioxidants. The first isomer is responsible for 6% and the

380

second isomer for 14% of total TEAC in C. incanus infusions.10 16 ACS Paragon Plus Environment

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381

From the results of this study, it can be concluded that most of the phenolic compounds in C.

382

incanus are present in the leafy parts. The dominant presence in leaves was generally

383

observed for most of the flavonoids, phenolic acids and the ellagitannins. Nevertheless for

384

costumer´s acceptance, a balanced flavor is of equal importance, besides the amount of

385

bioactive phenolic compounds. Infusions brewed from 100% (w/w) leaf material may be

386

rejected because of an overpowering `green odor´. However, there might be some unidentified

387

or under the actual brewing conditions non-extractable compounds in woody stem parts of C.

388

incanus. Varying extraction conditions, e.g. multiple maceration or use of other solvents can

389

lead to different results. In this context, it also has to be considered that short-term boiled hot

390

water extracts of the aerial parts have been traditionally used for the therapy of inflammatory

391

skin diseases, as well as for an improved wound healing, while hot water extracts boiled for a

392

longer time have been used as antidiarrhetics.3 For this reason product optimization regarding

393

stem and leaf contents of herbal tea mixtures as well as preparation guidelines are very

394

important for infusions containing high concentrations of bioactive phenolic compounds with

395

reference to the corresponding physiological property aimed at.

396 397

Influence of herbal particle size on the extractability of phenolic compounds. Besides

398

the question of the presence of phenolic compounds in leaves or woody stem material of C.

399

incanus, the different particle sizes of the herbal material may be also important for the

400

extractability of bioactive compounds during the brewing process. For investigating a

401

correlation between extractability of phenolic compounds and herbal particle size, exemplarily

402

C. incanus herbal tea sample 1 was separated by use of a laboratory sieving machine into six

403

fractions with different particle sizes (samples sv1-sv6). As obvious from Figure 1 B, all sieved

404

fractions contained recognizable parts of both, leaves and woody stem parts. Figure 3 shows 17 ACS Paragon Plus Environment


Page 18 of 34

405

the influence of the herbal particle size on the concentration of 22 phenolic compounds in the

406

resulting C. incanus infusions. This influence is demonstrated for the phenolic substance

407

classes of phenolic acids (A), flavan-3-ols and their dimers (B), myricetin glycosides (C),

408

quercetin glycosides (D), tiliroside isomers (E), and ellagitannins (F).

409

For most of the phenolic acids quantitated in the six measured C. incanus infusions brewed

410

from plant material with different average particle sizes from 0.3 mm up to 2.8 mm,

411

concentrations increased with decreasing particle size. For example, the concentration of

412

hydroxyferulic acid-4-O-rhamnoside increased gradually from 0.3 mg/100 mL infusion (sv1) up

413

to 1.1 mg/100 mL infusion (sv5). However, the lowest content of 0.2 mg/100 mL infusion for

414

this compound was detected for infusions brewed from 0.3 mm herbal material (sv6). A similar,

415

negative correlation between particle size and concentration of phenolic compound was

416

observed for gallic acid, methylgallate, and hydroxyferulic acid-5-O-rhamnoside. For ellagic

417

acid and ellagic acid-7-O-xyloside, the highest extraction yields were reached by use of herb

418

material with an average particle size of 0.3 mm (sv6). Additionally, ellagic acid-7-O-xyloside

419

only was present in quantitatable amounts in infusions brewed from herbal tea with particle

420

size less than 1.7 mm (sv3). Furthermore, hydroxyferuloyl-rhamnose reached the highest

421

concentration in the infusion resulting of C. incanus herbal tea with an average particle size of

422

1.2 mm (sv4). Altogether, the coefficients of correlation between particle size and

423

concentration of phenolic compound were very low for phenolic acids in C. incanus infusions.

424

However, all results for the extractability based on actual preparation recommendations of the

425

manufacturer. Longer extraction times or other extraction temperatures can lead to varying

426

concentrations of antioxidant phenolic substances in C. incanus infusions, as shown in a

427

recent study.10

428

Regarding the group of flavan-3-ols and dimers the highest extractability for gallocatechin

429

and catechin was achieved when using herbal tea consisting of an average particle size of 0.3 18 ACS Paragon Plus Environment

Page 19 of 34


430

mm (sv6). Altogether, gallocatechin concentrations ranged from 0.2 mg/100 mL infusion (sv1)

431

up to 0.9 mg/100 mL infusion (sv6) and for catechin from 0.5 mg/100 mL infusion (sv1) up to

432

1.8 mg/100 mL infusion (sv6). The correlation coefficients for the influence of the particle size

433

on the concentration of phenolic compound in the infusion were R² = 0.89 for gallocatechin

434

and R² = 0.93 for catechin. These results for the extractability of some phenolic compounds

435

were also supported by a study of Lang and Wai,40 who described the sample particle size

436

being a generally critical factor for a satisfactory extraction in herbal and natural products. In

437

the present work, catechin, which was detected in leaves but also in remarkably high contents

438

in woody stem parts, the extraction yield was almost 4-fold higher when extracting out of

439

herbal material with an average particle size of 0.3 mm (sv6) compared to a particle size of 2.8

440

mm (sv1). However, controversial results were reported for black tea infusions. Astill et al.41

441

measured higher catechin concentrations in infusions brewed from whole tea leaves compared

442

to infusions resulting from tea dust. In case of black tea, a higher degradation of catechin in

443

tea dust due to an enzymatic degradation (e.g. phenoloxidases) is the main explanation.41 In

444

C. incanus herbal teas with small particle sizes, such an endogenous degradation of phenolic

445

compounds seems to be negligible or may be overlaid by higher extraction yields.

446

A similar extractability was observed for the myricetin and quercetin glycosides. The

447

concentrations of myricitrin reached from 1.0 mg/100 mL infusion (sv1) up to 4.6 mg/100 mL

448

infusion (sv6) and for quercitrin from 0.3 mg/100 mL infusion (sv1) up to 1.0 mg/100 mL

449

infusion (sv6) with decreasing particle size. This means 5-fold higher concentrations for

450

myricitrin and 3-fold higher concentrations of quercitrin in C. incanus infusions brewed from

451

herb material with a comparatively small particle size. The coefficients of correlation between

452

particle size and concentration of phenolic compound were R² = 0.97 for myricitrin and R² =

453

0.96 for quercitrin.



Page 20 of 34

454

For the tiliroside isomers extractability seems to be different. For both isomers only an

455

approximately 1.5-fold higher amount was extracted from herbal tea with 0.3 mm average

456

particle size (sv6) compared to the infusion brewed from plant material with 2.8 mm herbal

457

particle size (sv1). From all these results it can be concluded that these acylated flavonol

458

glucosides were extracted very efficiently from plant material even at a rather large particle

459

size.

460

Regarding the extractability of the ellagitannins a strong influence of particle size on the

461

concentration in the corresponding infusions was observed for both hexahydroxydiphenoyl-

462

glucose isomers. For the first isomer at retention time of 27.1 min a 7-fold higher concentration

463

and for the second isomer at retention time of 28.7 min an 8-fold higher concentration was

464

yielded, when brewing the infusion from herbal tea with a particle size of 0.3 mm (sv6) instead

465

of using material with 2.8 mm average particle size (sv1). The absolute concentrations for the

466

second isomer reached from 0.3 mg/100 mL infusion (sv1) up to 2.5 mg/100 mL infusion (sv6).

467

Overall for ellagitannin rich C. incanus infusions, a small herbal particle size is decisive under

468

the actual brewing recommendations.

469

Altogether, the herbal particle size should be considered as an important factor for the

470

extractability of phenolic compounds in C. incanus infusions. For most of the bioactive

471

phenolic compounds quantitated in this work, infusions brewed from herbal tea with a

472

comparatively small particle size between 0.3 mm (sv6) and 0.8 mm (sv5) contained the

473

highest concentrations.

474 475 476 477

ACKNOWLEDGEMENT We thank Anna Knöpfle (Chemical Wood Technology, University of Hamburg, Germany) for excellent technical assistance.

478 20 ACS Paragon Plus Environment

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479

REFERENCES

480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525

(1) Attaguile, G.; Russo, A.; Campisi, A.; Savoca, F.; Acquaviva, R.; Ragusa, N.; Vanella, A. Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L.. Cell Biol. Toxicol. 2000, 16, 83-90. (2) Barrajón-Catalán, E.; Fernández-Arroyo, S.; Roldán, C.; Guillén, E.; Saura, D.; SeguraCarretero, A.; Micol, V. A systematic study of the polyphenolic composition of aqueous extracts deriving from several Cistus genus species: evolutionary relationship. Phytochem. Anal. 2011, 22, 303-312. (3) Petereit, F. Polyphenolische Inhaltsstoffe und Untersuchungen zur entzündungshemmenden Aktivität der traditionellen Arzneipflanze Cistus incanus L. (Cistaceae). Ph.D. Thesis, University of Münster, Germany, 1992. (4) Pott, R. Naturwirkstoffe aus Pflanzen. Biodiversitätsforschung. Chem. Unserer Zeit 2010, 44, 260-274. (5) Droebner, K.; Ehrhardt, C.; Poetter, A.; Ludwig, S.; Planz, O. CYSTUS052, a polyphenolrich plant extract, exerts anti-influenza virus activity in mice. Antiviral Res. 2007, 76, 1-10. (6) Ehrhardt, C.; Hrincius, E. R.; Korte, V.; Mazur, I.; Droebner, K.; Poetter, A.; Dreschers, S.; Schmolke, M.; Planz, O.; Ludwig, S. A polyphenol rich plant extract, CYSTUS052, exerts anti influenza virus activity in cell culture without toxic side effects or the tendency to induce viral resistance. Antiviral Res. 2007, 76, 38-47. (7) Droebner, K.; Haasbach, E.; Mueller, C.; Ludwig, S.; Planz, P. The polyphenol rich plant extract CYSTUS052 is highly effective against H5N1 and pandemic H1N1v influenza A viruses. Influenza Other Respir. Viruses 2011, 5, 230-251. (8) Kalus, U.; Grigorov, A.; Kadecki, O.; Jansen, J.-P.; Kiesewetter, H.; Radtke, H. Cistus incanus (CYSTUS052) for treating patients with infection of the upper respiratory tract A prospective, randomised, placebo-controlled clinical study. Antiviral Res. 2009, 84, 267271. (9) Hannig, C.; Spitzmüller, B.; Al-Ahmad, A.; Hannig, M. Effects of Cistus-tea on bacterial colonization and enzyme activities of the in situ pellicle. J. Dent. 2008, 36, 540-545. (10) Riehle, P.; Vollmer, M.; Rohn, S. Phenolic compounds in Cistus incanus herbal infusions Antioxidant capacity and thermal stability during the brewing process. Food Res. Int. 2013, 53, 891-899. (11) Scalbert, A.; Manach, C.; Morand, C.; Rémésy, C.; Jiménez, L. Dietary polyphenols and the prevention of diseases. Crit. Rev. Food Sci. Nutr. 2005, 45, 287-306. (12) Pomponio, R.; Gotti, R.; Santagati, N.; Cavrini, V. Analysis of catechins in extracts of Cistus species by microemulsion electrokinetic chromatography. J. Chromatogr. A 2003, 990, 215-223. (13) Arts, I. C. W.; Hollmann, P. C. H.; Feskens, E. J. M.; De Mesquita, H. B. B.; Kromhout, D. Catechin intake might explain the inverse relation between tea consumption and ischemic heart disease: the Zutphen Elderly Study. Am. J. Clin. Nutr. 2001, 74, 227-232. (14) Neveu, V.; Perez-Jiménez, J.; Vos, F.; Crespy, V.; Du Chaffaut, L.; Mennen, L.; Knox, C.; Eisner, R.; Cruz, J.; Wishart, D.; Scalbert, A. Phenol-Explorer: an online comprehensive database on polyphenol contents in foods. Database 2010, 1-9. (15) Rothwell, J. A.; Urpi-Sarda, M.; Boto-Ordoñez, M.; Knox, C.; Llorach, R.; Eisner, R.; Cruz, J.; Neveu, V.; Wishart, D.; Manach, C.; Andres-Lacueva, C.; Scalbert, A. Phenol-Explorer 2.0: a major update of the Phenol-Explorer database integrating data on polyphenol metabolism and pharmacokinetics in humans and experimental animals. Database 2012, 1-8. 21 ACS Paragon Plus Environment


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(16) Rothwell, J. A.; Pérez-Jiménez, J.; Neveu, V.; Medina-Ramon, A.; M'Hiri, N.; Garcia Lobato, P.; Manach, C.; Knox, K.; Eisner, R.; Wishart, D.; Scalbert, A. Phenol-Explorer 3.0: a major update of the Phenol-Explorer database to incorporate data on the effects of food processing on polyphenol content. Submitted 2013. (17) Goto, T.; Teraminami, A.; Lee, J.-Y.; Ohyama, K.; Funakoshi, K.; Kim, Y.-I.; Hirai, S.; Uemura, T.; Yu, R.; Takahashi, N.; Kawada, T. Tiliroside, a glycosidic flavonoid, ameliorates obesity-induced metabolic disorders via activation of adiponectin signaling followed by enhancement of fatty acid oxidation in liver and skeletal muscle in obesediabetic mice. J. Nutr. Biochem. 2012, 23, 768-776. (18) Silva, G. C.; Pereira, A. C.; Rezende, B. A.; Da Silva, J. F. P.; Cruz, J. S.; De Souza, M. de F. V.; Gomes, R. A.; Teles, Y. C. F.; Cortes, S. F.; Lemos, V. S. Mechanism of antihypertensive and vasorelaxant effects of the flavonoid tiliroside in resistance ateries. Planta Med. 2013, 79, 1003-1008. (19) Chaves, N.; Rios, J. J.; Gutierrez, C.; Escudero, J. C.; Olias, J. M. Analysis of secreted flavonoids of Cistus ladanifer L. by high-performance liquid chromatography particle beam mass spectrometry. J. Chromatogr. A 1998, 799, 111-115. (20) Saracini, E.; Tattini, M.; Traversi, M. L.; Vincieri, F. F.; Pinelli, P. Simultaneous LC-DAD and LC-MS determination of ellagitannins, flavonoid glycosides, and acyl-glycosyl flavonoids in Cistus salvifolius L. leaves. Chromatographia 2005, 62, 245-249. (21) Santagati, N. A.; Salerno, L.; Attaguile, G.; Savoca, F.; Ronsisvalle, G. Simultaneous determination of catechins, rutin, and gallic acid in Cistus species extracts by HPLC with diode array detection. J. Chromatogr. Sci. 2008, 46, 150-156. (22) Fernández-Arroyo, S.; Barrajón-Catalán, E.; Micol, V.; Segura-Carretero, A.; FernándezGutiérrez, A. High-performance liquid chromatography with diode array detection coupled to electrospray time-of-flight and ion-trap tandem mass spectrometry to identify phenolic compounds from a Cistus ladanifer aqueous extract. Phytochem. Anal. 2009, 21, 307313. (23) Qa'Dan, F.; Petereit, F.; Mansoor, K.; Nahrstedt, A. Antioxidant oligomeric proanthocyanidins from Cistus salvifolius. Nat. Prod. Res. 2006, 20, 1216-1224. (24) Breitfellner, F.; Solar, S.; Sontag, G. Effect of gamma-irradiation on phenolic acids in strawberries. J. Food Sci. 2002, 67, 517-521. (25) Willför, S.; Pranovich, A.; Tamminen, T.; Puls, J.; Laine, C.; Suurnäkki, A.; Saake, B.; Uotila, K.; Simolin, H.; Hemming, J.; Holmbom, B. Carbohydrate analysis of plant materials with uronic acid-containing polysaccharides - A comparison between different hydrolysis and subsequent chromatographic analytical techniques. Ind. Crops Prod. 2009, 29, 571-580. (26) Pott, R.; Hüppe, J. 9 Anpassungen der Pflanzen. In Spezielle Geobotanik: Pflanze – Klima – Boden; Springer-Verlag: Berlin Heidelberg, Germany, 2007, 232-282. (27) Aronne, G.; De Micco, V. Seasonal dimorphism in the Mediterranean Cistus incanus L. subsp. incanus. Ann. Bot. 2001, 87, 789-794. (28) Christodoulakis, N. S.; Georgoudi, M.; Fasseas, C. Leaf structure of Cistus creticus L. (Rock Rose), a medicinal plant widely used in folk remedies since ancient times. J. Herbs, Spices Med. Plants 2014, 20, 103-114. (29) Hernández, I.; Alegre, L.; Munné-Bosch, S. Drought-induced changes in flavonoids and other low molecular weight antioxidants in Cistus clusii grown under Mediterranean field conditions. Tree Physiol. 2004, 4, 1303-1311. (30) Meyer-Buchtela, E. Tee-Rezepturen: Ein Handbuch für Apotheker und Ärzte, mit 2. Ergänzungslieferung; Deutscher Apotheker Verlag: Stuttgart, Germany, 2001. 22 ACS Paragon Plus Environment

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574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602


(31) Souquet, J.-M.; Labarbe, B.; Le Guernevé, C.; Cheynier, V.; Moutounet, M. Phenolic composition of grape stems. J. Agric. Food Chem. 2000, 48, 1076-1080. (32) Lavola, A.; Karjalainen, R.; Julkunen-Tiitto, R. Bioactive polyphenols in leaves, stems, and berries of Saskatoon (Amelanchier alnifolia Nutt.) cultivars. J. Agric. Food Chem. 2012, 60, 1020-1027. (33) Jürgenliemk, G.; Petereit, F.; Nahrstedt, A. Flavan-3-ols and procyanidins from the bark of Salix purpurea L.. Pharmazie 2007, 62, 231-234. (34) Harris, C. U.; Burt, A. J.; Saleem, A.; Maile, P.; Martineau, L. C.; Haddad, P. S.; Bennett, S. A. L.; Aanason, J. T. A single HPLC-PAD-APCI/MS method for the quantitative comparison of phenolic compounds found in leaf, stem, root and fruit extracts of Vaccinium angustifolium. Phytochem. Anal. 2007, 18, 161-169. (35) Tian, C.; Wang, M.; Liu, X.; Wang, H.; Zhao, C. HPLC quantification of nine chemical constituents from the five parts of Abutilon theophrasti Medic. J. Chromatogr. Sci. 2014, 52, 258-263. (36) Peleg, H.; Gacon, K.; Schlich, P.; Noble, A. C. Bitterness and astringency of flavan-3-ol monomers, dimers and trimers. J. Agric. Food Chem. 1999, 79, 1123-1128. (37) Scharbert, S.; Hofmann, T. Molecular definition of black tea taste by means of quantitative studies, taste reconstitution, and omission experiments. J. Agric. Food Chem. 2005, 53, 5377-5384. (38) Hatanaka, A. The biogeneration of green odor by green leaves. Phytochemistry 1993, 34, 1201-1218. (39) Hongsoongnern, P.; Chambers IV, E. A lexicon for green odor or flavor and characteristics of chemicals associated with green. J. Sens. Stud. 2008, 23, 205-221. (40) Lang, Q.; Wai, C. M. Supercritical fluid extraction in herbal and natural product studies - a practical review. Talanta 2001, 53, 771-782. (41) Astill, C.; Birch, M. R.; Dacombe, C.; Humphrey, P. G.; Martin, P. T. Factors affecting the caffeine and polyphenol contents of black and green tea infusions. J. Agric. Food Chem. 2001, 49, 5340-5347.



Page 24 of 34

603

Figure captions

604

Figure 1: A) Three manually sorted C. incanus herbal teas (out of sample 1). Plant part

605

composition of samples: st1) 0% (w/w) leaves and 100% (w/w) woody stems, st3) 50%

606

(w/w) leaves and 50% (w/w) woody stems, st5) 100% (w/w) leaves and 0% (w/w)

607

woody stems. B) Sieved C. incanus herbal teas (out of sample 1) containing leaves and

608

woody stem parts. Average particle sizes of herb material: sv1) 2.8 mm, sv2) 2.3 mm,

609

sv3) 1.7 mm, sv4) 1.2 mm, sv5) 0.8 mm and sv6) 0.3 mm.

610

Figure 2: Contents of 22 phenolic compounds in five C. incanus infusions brewed from herbal

611

mixtures (out of sample 1) with leaf contents of 0% (w/w), 25% (w/w), 50% (w/w), 75%

612

(w/w) and 100% (w/w) and corresponding woody stem contents of 100% (w/w), 75%

613

(w/w), 50% (w/w) and 0% (w/w). A) phenolic acids; B) flavan-3-ols and -dimers; C)

614

myricetin glycosides; D) quercetin glycosides; E) tiliroside isomers and F) ellagitannins.

615

For compound no. refer to Table 2.

616

Figure 3: Contents of 22 phenolic compounds in six C. incanus infusions brewed from herbal

617

material (out of sample 1) with average particle size of 2.8 mm, 2.3 mm, 1.7 mm, 1.2

618

mm, 0.8 mm and 0.3 mm, all concluding recognizable leaf and stem parts (Figure 1 B).

619

A) phenolic acids; B) flavan-3-ols and -dimers; C) myricetin glycosides; D) quercetin

620

glycosides; E) tiliroside isomers and F) ellagitannins. For compound no. refer to Table

621

2.

622


Page 25 of 34


623

Table 1. Range of average leaf and stem particle sizes, stem diameters and country of origin (according to manufacturer information)

624

of the four analyzed C. incanus herbal teas (sample 1-4).

compound name particle size [mm] diameter [mm] country of origin

sample 1

sample 2

sample 3

sample 4

leaves

stems

leaves

stems

leaves

stems

leaves

stems

1.0-7.0

2.0-15.0

1.0-7.0

4.0-15.0

1.0-5.0

3.0-10.0

1.0-4.0

3.0-7.0

0.5-4.0 Greece

0.5-3.0 Greece

0.5-3.0 unknown

0.5-1.0 Turkey

625



Page 26 of 34

626

Table 2. Quantitated phenolic compounds in C. incanus infusions: Compound no., MS-data, retention times and corresponding SPE

627

eluates.

-

no.

compound name

[M-H] [m/z]

MS/MS fragments (relative intensity %) [m/z]

Rt [min]

SPE eluate

1

gallic acid

169

125

(100)

2.9

MeOH

2

gallocatechin

305

287

(4)

261

(35)

221

(82)

219

(90)

179

(100)

5.6

MeOH

3

gallocatechin-(4α-8)-catechin or catechin-(4α-8)-gallocatechin

593

575

(3)

467

(10)

425

(100)

407

(28)

289

(14)

6.6

MeOH

4

catechin

289

271

(3)

245

(100)

205

(38)

179

(14)

11.6

MeOH

5

myricetin-3-O-galactoside

479

461

(1)

317

(87)

316

(94)

271

(2)

179

(4)

19.5

MeOH

6

myricetin-3-O-glucoside

479

461

(3)

317

(94)

316

(98)

271

(1)

179

(4)

19.8

MeOH

7

myricetin-O-xyloside

449

431

(4)

317

(51)

316

(100)

271

(1)

179

(4)

21.9

MeOH

8

myricitrin

463

445

(1)

317

(83)

316

(100)

179

(4)

23.0

MeOH

9

quercetin-3-O-galactoside

463

445

(1)

301

(100)

179

(3)

23.7

MeOH

10

quercetin-3-O-glucoside

463

445

(1)

301

(100)

179

(2)

23.2

MeOH

11

quercetin-O-xyloside

433

301

(100)

179

(2)

26.6

MeOH

12

quercitrin

447

429

(1)

301

(100)

179

(1)

28.3

MeOH

13

trans-tiliroside

593

447

(13)

307

(7)

285

(100)

257

(2)

41.1

MeOH

14

cis-tiliroside

593

447

(13)

307

(7)

285

(100)

257

(2)

42.5

MeOH

15

methylgallate

183

139

(100)

2.6

MeOH/NH3

16

hydroxyferulic acid-5-O-rhamnoside

355

337

(20)

209

(24)

191

(100)

173

(3)

147

(4)

15.3

MeOH/NH3

17


355

337

(14)

209

(24)

191

(100)

173

(3)

147

(2)

17.7

MeOH/NH3

18

hydroxyferuloyl-rhamnose

355

337

(17)

209

(22)

191

(100)

173

(3)

147

(3)

22.3

MeOH/NH3

19

HHDP-glucose isomer

482

464

(3)

450

(33)

406

(3)

300

(100)

271

(5)

27.1

MeOH/NH3

20

HHDP-glucose isomer

481

463

(20)

449

(100)

405

(16)

299

(54)

270

(7)

28.7

MeOH/NH3

21

ellagic acid-7-O-xyloside

433

301

(100)

35.8

MeOH/NH3


Page 27 of 34

22

628 629


ellagic acid

301

257

(11)

229

(7)

185

(1)

33.8

MeOH/NH3

Rt: Retention time during LC-DAD analysis; MeOH: Methanolic SPE eluate; MeOH/NH3: Ammonia containing methanolic SPE eluate; HHDP-glucose: hexahydroxydiphenoyl-glucose

630



Page 28 of 34

631

Table 3. Total ASE extract content, hydrolysis residues, contents of glucose, xylose and sum of glucose and xylose as well as

632

cellulose contents of exemplarily out of sample 1 sorted C. incanus herbal teas (no. st1-st5). Glucose values of the starch test are

633

shown for samples no. st1 and st5.

sample no.

leaf parts

stem parts

hydrolysis residue [%]

glucose

xylose

[% w/w]

total ASE extract content [%]

cellulose

[%]

glucose + xylose [%]

[%]

glucose (from starch) [%]

[% w/w]

[%]

st1

0

100

2

26

34

21

55

30

1

st2

25

75

7

26

29

17

46

26

st3

50

50

12

28

24

13

37

21

st4

75

25

18

31

19

9

28

17

st5

100

0

22

35

14

5

19

12

1

634


Page 29 of 34


635

Table 4. Concentration [mg/100 mL] of phenolic compounds in four samples of C. incanus infusions brewed from manually sorted

636

herbal tea including 100 % (w/w) leaves and 100 % (w/w) stems.

no.

compound name

1

sample 1

sample 2

sample 3

sample 4

leaves

stems

leaves

stems

leaves

stems

leaves

stems

gallic acid

1.1±0.03

0.1±0.00

1.1±0.02

0.2±0.01

1.1±0.01

0.3±0.01

1.2±0.01

0.4±0.03

2

gallocatechin

1.7±0.17

nd

1.6±0.06

0.2±0.02

1.0±0.10

0.2±0.02

0.7±0.04

nd

3

gallocatechin-(4α-8)-catechin or catechin-(4α-8)-gallocatechin

0.8±0.06

nd

0.9±0.10

0.2±0.01

0.7±0.02

0.2±0.02

0.8±0.08

0.3±0.02

4

catechin

0.8±0.04

0.5±0.05

1.2±0.06

1.1±0.07

0.6±0.06

0.9±0.04

0.4±0.02

0.3±0.02

5

myricetin-3-O-galactoside

3.2±0.16

nd

1.4±0.06

0.1±0.00

2.1±0.03

0.1±0.01

1.4±0.03

0.1±0.01

6

myricetin-3-O-glucoside

0.4±0.03

nd

0.2±0.01

nd

0.2±0.01

nd

0.2±0.01

nd

7

myricetin-O-xyloside

1.1±0.11

nd

0.4±0.01

nd

0.7±0.03

nq

0.4±0.02

nq

8

myricitrin

8.4±0.22

nd

5.8±0.12

0.1±0.02

4.7±0.09

0.2±0.01

8.4±0.19

0.5±0.01

9

quercetin-3-O-galactoside

0.8±0.03

nd

0.6±0.03

0.1±0.01

1.3±0.06

0.1±0.01

0.5±0.02

0.1±0.01.1

10

quercetin-3-O-glucoside

0.1±0.01

nd

0.1±0.01

nq

0.2±0.02

nq

0.1±0.01

nq

11

quercetin-O-xyloside

0.4±0.03

nd

0.4±0.17

nq

0.4±0.18

0.1±0.01

0.3±0.02

nq

12

quercitrin

1.6±0.04

nd

1.0±0.05

0.1±0.01

0.8±0.01

0.2±0.01

1.8±0.04

0.1±0.01

13

trans-tiliroside

0.9±0.04

nd

0.9±0.05

0.1±0.01

0.8±0.02

0.2±0.01

0.6±0.03

0.1±0.01

14

cis-tiliroside

0.3±0.01

nd

0.3±0.00

nq

0.3±0.01

nq

0.2±0.01

nq

15

methylgallate

0.2±0.01

0.1±0.01

0.3±0.02

0.1±0.00

0.5±0.02

0.2±0.00

0.3±0.03

0.2±0.00

16


1.0±0.06

nd

2.6±0.25

0.1±0.04

1.7±0.06

0.4±0.04

1.9±0.11

0.2±0.06

17


2.2±0.18

nd

2.6±0.15

0.4±0.02

2.6±0.10

0.9±0.09

1.9±0.11

0.5±0.06

18

hydroxyferuloyl-rhamnose

0.6±0.04

nd

1.0±0.13

nq

1.3±0.08

0.2±0.03

1.1±0.11

nq

19

HHDP-glucose isomer

0.5±0.03

nd

0.7±0.15

nd

0.5±0.01

nd

0.2±0.02

nd

20

HHDP-glucose isomer

0.9±0.07

nd

2.0±0.10

0.1±0.02

2.1±0.05

0.3±0.02

0.5±0.05

nd

21

ellagic acid-7-O-xyloside

0.1±0.03

nd

0.1±0.03

nd

0.1±0.01

nd

0.1±0.02

nd

22

ellagic acid

0.7±0.05

nd

0.4±0.01

0.1±0.01

0.4±0.02

0.1±0.01

0.2±0.02

0.1±0.01



637 638

Page 30 of 34

Compound no.; nd: not detectable; nq: not quantitatable; HHDP-glucose: hexahydroxydiphenoyl-glucose.


Page 31 of 34

639 640


Figure 1:



642 643

Page 32 of 34

Figure 2:


Page 33 of 34

645 646


Figure 3:



648

Page 34 of 34

Table of contents (TOC):

649

650


Influence of the Leaf Content and Herbal Particle Size on the

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