Analysis of Phenolic Compounds in Rooibos Tea (Aspalathus linearis

Oct 24, 2017 - Tea samples from 17 populations of “wild tea” ecotypes Aspalathus linearis (rooibos tea) and 2 populations of Aspalathus pendula we...
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Analysis of phenolic compounds in rooibos tea (Aspalathus linearis) with a comparison of flavonoid-based compounds in natural populations of plants from different regions Marietjie Stander, Ben-Erik Van Wyk, Malcolm John C. Taylor, and Helen Selma Long J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03942 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Analysis of phenolic compounds in rooibos tea (Aspalathus linearis) with a comparison of flavonoid-based compounds in natural populations of plants from different regions Maria A. Stander*,a,c, Ben-Erik Van Wykb, Malcolm J.C. Taylorc, Helen S. Longb a

Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland 7600,

South Africa. b

Department of Botany and Plant Biotechnology, University of Johannesburg, P.O. Box 524,

Auckland Park 2006, Johannesburg, South Africa. c

Mass Spectrometry Unit, Central Analytical Facility, University of Stellenbosch, Private Bag

X1, Matieland 7600, South Africa.

*Corresponding author,(Tel: +27 21 808 5825; Fax: +27 21 808 5863; E-mail:[email protected]);

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ABSTRACT: Tea samples from 17 populations of “wild tea” ecotypes Aspalathus linearis

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(rooibos tea), and two populations of A. pendula were analyzed. Recent advances in column

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technology together with high-resolution mass spectrometry were applied to improve resolution,

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facilitating the identification of several new compounds, as well as grouping of the wild tea

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ecotypes according to their chemical composition. The collisional cross section (CCS) data

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obtained from ion mobility-mass spectrometry is reported for the flavonoids in rooibos for the

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first time. Enzyme pathways for the synthesis of the unique flavonoids found in rooibos tea are

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also proposed. Aspalathus linearis and A. pendula produce similar combinations of main

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phenolic compounds, with no diagnostically different discontinuities between populations or

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species. Northern resprouters (Gifberg and Nieuwoudtville) contain higher PPAG levels whilst

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teas from Wupperthal and surrounding areas were found to contain unique dihydrochalcones

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(phloridzin and a sieboldin analog) are reported here for the first time.

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KEYWORDS: Aspalathus linearis, rooibos tea, flavonoids, ecotypes, wild tea profiling, liquid chromatography-mass spectrometry

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Introduction

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The aim of this study was a comprehensive characterization of the phenolic constituents, not

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only of the commercial (cultivated) form of rooibos tea but of the entire species complex known

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botanically known as Aspalathus linearis (Burm.f.) Dahlg. The phenolic profile of commercial

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rooibos tea has been thoroughly investigated, but the chemical compositions of numerous wild

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tea types belonging to this species complex have remained poorly explored.

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Aspalathus linearis exists as at least eight distinct sub-forms or ecotypes that have developed

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due to geographic isolation and various mutations conferring survival advantage in response to

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differing environments, especially exposure to fire1-3. Four of the ecotypes are reseeders (also

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called seeders) – single-stemmed plants that are destroyed by fire and that recruit from seeds in

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the soil seed bank, whilst four others are resprouters (also called sprouters) – multi-stemmed

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plants arising from a persistent underground lignotuber, which regenerates (coppices) after

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fire3,4. The ecotypes are also ecologically distinct, with some ecotypes preferring habitats at

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higher elevations (>400–600m), higher rainfall sites in the southern areas of the range, or lower

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rainfall sites in the northern areas2. The eight main geographical variants or ecotypes have been

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described and illustrated by Van Wyk and Gorelik3 and comprise (1) southern sprouter; (2) grey

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sprouter; (3) northern sprouter; (4) Nieuwoudtville sprouter; (5) red type (seeder); (6) black type

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(seeder); (7) Wupperthal type (seeder) and the (8) tree type (seeder).

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Commercial rooibos tea, known as the Red type or Rocklands type, is a seeder with an upright

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growth form that was selected and developed as a new crop plant by Nortier in the early part of

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the 20th century (ca. 1920–1930). The selection criteria were taste, tea colour, growth rate, and

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seed production2. Initially seeds were collected from the Pakhuis region and scarification of the

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seed coat was found to be necessary for successful germination. Whilst the Red type is farmed

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extensively and represents a major industry in the Cedarberg region, there is also significant

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small scale wild-harvesting (wild-crafting) of various wild types, especially in the Wupperthal

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and Nieuwoudtville areas of the A. linearis range1. The main flavonoids in the leaves of rooibos

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have been identified as aspalathin, nothofagin, orientin, isoorientin, vitexin, isovitexin,

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isoquercetin, quercetin and rutin5-8. De Beer et al. 9 found that aspalathin and nothofagin

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production in unfermented rooibos show slight seasonal variations, with the highest

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concentration in mid-spring to early summer. The other flavonoids in the study did not vary

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significantly. This agrees with a study by Joubert et al. 10 where 209 commercial fermented teas

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of the Red type (Rocklands type) from different production regions were analysed. It was found

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that the phenolic and phenylpropenoic acid glucoside (PPAG) content were similar with slight

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differences between production years. PPAG is another unique compound only found in rooibos.

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The goal of the current study was to re-examine the phenolic profile of the wild type teas using

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high resolution chromatographic and mass spectrometric systems in order to gain a more

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profound understanding of the phenolic diversity in A. linearis. This knowledge is not only of

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academic interest but may also have practical value in developing or improving quality control

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protocols and in identifying various chemotypes that can potentially be developed and

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commercialised as unique geographical indications with novel flavours and health benefits.

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Materials and methods

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Sampling

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In an attempt to overcome problems due to sample non-homogeneity, representative samples

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were taken from 10 randomly selected individual plants of each of the 17 wild-growing natural

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populations of rooibos tea (Aspalathus linearis), together with two samples from the closely

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related A. pendula (Table 1). These samples were combined and processed using the standard

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methods for processing rooibos tea. They were cut, mixed, moisturised, bruised, oxidised in

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sunlight and then rapidly dried. The vouchers specimens have been deposited in the University

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of Johannesburg Herbarium (JRAU). Figure 1 depicts the locations of the various tea

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populations that were sampled.

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Extraction

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Approximately 2 grams of dry tea was ground in a pestle and mortar and extracted with 50%

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methanol in water containing 1% formic acid (15 ml) in a 50 mL polypropylene centrifuge tube

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by soaking it overnight, followed by extraction in an ultrasonic bath (0.5 Hz, Integral systems,

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RSA) for 60 minutes at room temperature. The extracts were centrifuged (Hermle Z160m, 3000

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x g for 5 minutes) and transferred to vials.

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Standards

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Standards were obtained from Sigma-Aldrich and from a kind donation by D. De Beer of the

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Agricultural Research Council (ARC). Stock solutions were prepared quantitatively in cocktails

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ranging from 1 to 100 µg/mL in concentration. Four different cocktails were prepared at each

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level to enable isomers and compounds with similar elemental formulae to be distinguished. The

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solvent used for preparation of the cocktails was 50% methanol in water containing 1% formic

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acid, i.e., the same as that used for extraction of the tea samples.

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LCMS analysis

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A Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS) connected to a

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Waters Acquity ultra performance liquid chromatograph (UPLC) (Waters, Milford, MA, USA)

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was used for high resolution UPLC-MS analysis. Electrospray ionization was used in negative

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mode with a cone voltage of 15 V, desolvation temperature of 275 °C, desolvation gas at 650

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L/hr and the rest of the MS settings optimized for best resolution and sensitivity. Data were

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acquired by scanning from m/z 150 to 1500 in resolution mode as well as in MSE mode. In MSE

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mode two channels of MS data were acquired, one at a low collision energy (4 V) and the second

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using a collision energy ramp (40 to 100 V) to obtain fragmentation data as well. Leucine

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enkaphalin was used as lock mass (reference mass) for accurate mass determination and the

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instrument was calibrated with sodium formate. Separation was achieved on a Waters HSS T3,

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2.1 x 100 mm, 1.7 µm column. An injection volume of 2 µL was used and the mobile phase

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consisted of 0.1% formic acid (Solvent A) and acetonitrile containing 0.1 % formic acid as

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solvent B. The gradient started at 100% solvent A for 1 minute and changed to 28 % B over 22

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minutes in a linear way. It then went to 40% B over 50 seconds and a wash step of 1.5 minutes

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at 100% B, followed by re-equilibration to initial conditions for 4 minutes. The flow rate was

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0.3 mL/min and the column temperature was maintained at 55 ºC.

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Ion mobility data was obtained using the same UPLC gradient and column as above and IMS

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Wave velocity was set at 332 m/s and wave height at 20.2 V. Polyalanine was used for the

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calibration and calculations and the rest of the settings were according to Rautenbach et al.

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(2017).11

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Results and discussion Sample preparation and Extraction

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Samples were fermented to compare the compounds found with those present in commercial

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rooibos tea (Red type). Walters et al. 12 have done a large amount of work to optimize the

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extraction of rooibos tea. An extraction solvent of 50% methanol with 1% formic was used in

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this study since it showed a slightly higher extraction efficiency for the more polar, early eluting

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molecules (as well as PPAG and aspalathin) than the 40% acetonitrile solvent of Walters12. This

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solvent is also more environmentally friendly than acetonitrile. The extraction solvent of Walters

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was better for the more non-polar and later eluting molecules (when using sonication) (Figure

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1S).

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LCMS method

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There are a few critical separations that have to be achieved when analyzing the phenolic

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compounds in rooibos using liquid chromatography (Walters et al. 12), since several compounds

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are present with the same nominal mass. Figure 2 shows the extracted mass chromatograms at

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m/z 447, 449 and 609 representing separations between orientin/isorientin (447), R/S-eriodictyol-

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6-C-glucopyranoside and R/S-eriodictyol-8-C-glucopyranoside (449), quercetin-3-O-

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robinobioside and rutin (609). This separation improves if the photo diode array (PDA) detector

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is taken out of the system since peak broadening caused by the additional path length and flow

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cell is eliminated (Figure 2S). The aim of the study was to compare the relative concentrations

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of the flavonoids to each other in the various tea samples. It was noted that the extracted mass

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chromatograms of the standards contained in our cocktails showed similar responses in ESI

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negative mode whilst the peak areas produced by the UV detector were more variable due to the

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compounds exhibiting different UV maxima and absorbance at set wavelengths (Figure 3S).

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Integration of the peaks in the extracted mass chromatograms [M-H]-1 for the standards (Figure

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3S) for example vicenin-2 (compound 22), iso-orientin (30), vitexin (36), isoquercitrin (40),

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kaempferol glucoside (47) and phloridzin (51) resulted in peak areas that were very close in

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magnitude, showing a relative standard deviation (RSD) of only 4.5% at an injected

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concentration of 100 µg/mL. It was thus decided to use rutin as reference calibrant for

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compounds where no standards were available and to quantify compounds based on the areas of

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their extracted mass chromatograms, since the UV response was shown to be less selective and

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less sensitive (Figure 4S). A typical commercial rooibos tea sample was used as a control

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sample at the beginning and end of the run. The reproducibility of the extraction method was

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tested by extracting the same commercial tea sample five times and relative standard deviations

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(RSDs) between 6 and 13% were observed for the six main peaks (data not shown). The

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intraday variation was determined by reinjecting the same sample extracts a week later. The

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variation was between 16.5 and 29%, with aspalathin at 16.5 and the eriodictyol-C-b-D-

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glucopyranosides at the higher end. This is due to the instability of flavonoids in solution, all

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samples was analyzed for quantitative purposes in a 24 hour period.

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Table 2 contains the compounds that were detected in the rooibos tea samples. Many of these

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constituents were previously identified by other authors (Joubert5, Krafczyk et al.6, Iswaldi et al.

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7

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fragments, UV maxima and relative retention times reported in these previous papers.

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The collisional cross section (CCS) values were validated against those of Gonzales et al. 13. All

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of the CCS values that were also measured in the paper of Gonzales et al. 13 corresponded with

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the values in this paper. For example, a value of 230.6 Ȧ2 was reported for rutin; our value was

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230.8 Ȧ2. The highest deviation from Gonzales et al. 13 was found in the smaller molecules, but

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it was still within 2.2% of what they reported. There are a few compounds in Table 2 that has

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more than one CCS value. Figure 3 shows an extracted mass chromatograms (C) of m/z 449,

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447, 451 that corresponds to compounds 20,23,26 and 27 the eriodictyol glucopyranosides (m/z

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449) as well as m/z 451 (aspalathin) and 447 (orientin, isoorientin). It also shows the 2D ion

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mobilogram on top showing the peaks corresponding to the eriodictyol glucopyranosides having

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two spots (B) and a mobilogram of compound 20 showing two peaks at two drift times.

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Something similar was observed for chlorogenic acid. Kuhnert et al., 2015 reported this

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phenomenon as a differentiation of the prototropic ions, which means that the ions can carry their

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charge on two different positions that results in different shapes of the molecules (ions) and thus

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different collisional cross sections 14.

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Interestingly, no naringenin was detected in any of the samples, neither as the chalcone nor as the

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flavanone. This indicates that naringenin, which represents the starting point of the classical

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flavonoid pathway, is rapidly converted either to eriodictyol or to one of the dihydroflavonols

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(Figure 4). Another possibility is that it may not be formed at all due the dominance of the

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alternate pathway involving the production of phloretin via the carbon double bond reductase

and Walters et al.12) and were identified in this study according to their accurate mass, MSMS

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(CDBR) enzyme (Figure 5). A number of new compounds were detected, with tentative

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identifications being made based on the accurate mass determinations and comparison of the

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elemental compositions to databases such as Metlin and Chemspider. If possible, further

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confirmation was achieved by injecting reference standards. These compounds include 3-O-

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caffeoylquinic acid (compound 15, chlorogenic acid), catechin (compound 13), citric acid

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(compound 1), dihydroxy benzoic acid (compound 5), epicatechin (compound 19), p-coumaric

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acid (compound 29) and hesperetin (compound 58) (Figure 5S). All except citric acid were

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minor constituents. Phloretin-2-β-D-glucoside (compound 51, phloridzin) and phloretin-4-O-

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glucoside (compound 54, trilobatin) are newly identified compounds that were present in some

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of the tea samples and these determinations were confirmed with reference standards. A new

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dihydrochalcone glycoside (compound 44) that has the same accurate mass (m/z 451.1237, UV

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maximum (284 nm) and MS fragment ions (m/z 289,167,123) as sieboldin was also detected in

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some of the samples (Figure 6S). The retention time and the CCS value (Table 2) did not

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correspond to the sieboldin standard and it was tentatively identified as an isomer of sieboldin.

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Since sieboldin is an O-glucoside of luteolin dihydrochalcone, this compound is likely to also be

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a glucoside with the glucose attached at another position, most probably at the same position as

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in phloridzin, which is the 2’-β-D-glucoside (Figure 6).

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In some instances where authentic standards were not available in our laboratory, the tentative

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identifications were made from accurate mass and elemental composition with further

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confirmations using MS fragmentation spectra from literature. Compounds identified in this way

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were α-1-caffeoylglucose (compound 11) and piscidic acid (compound 6, Figure 7S). α-1-

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Caffeoylglucose (M-H of 341.0873) gave a fragment at m/z 179, indicative of caffeic acid and

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also a neutral loss of 162, characteristic of the loss of a hexose, its secondary fragment ion of m/z

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135 indicated that the position of the glucose is most probably at position 1 according to Jaiswal

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et al.15. Piscidic acid was confirmed by its fragment ions m/z 165,193,179, and its accurate mass.

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Furthermore, it eluted early in the run, similar to what Mata et al. 16 has found in Opuntia juice

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(Figure 7S). Piscidic acid belongs to the family of phenylpyruvic acid derivatives.

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The compound (52) that eluted at 20.77 minutes had an accurate mass of 493.1331 and fragment

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ions similar to those of aspalathin (which eluted earlier). It had a mass difference of m/z 42,

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characteristic of an acetyl group. The compound, was tentatively identified as an acetylated form

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of aspalathin, most probably an acetylglucoside.

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The compound (46) that eluted at 18.9 minutes had a molecular ion of m/z 469.1345 (C21H25O12)

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with the same fragments as PPAG (M-H: C15H17O8) (325,163,119) and was thus tentatively

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identified as a PPAG derivative (Figure 8S). The compound (18) that eluted at 13.0 minutes had

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a molecular ion of m/z 327.107 (M-H: C15H19O8) and therefore differs with two protons from

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PPAG, as does its first fragment (165,147,103). It has a similar UV spectrum to that of PPAG

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and was thus tentatively identified as a PPAG derivative, most probably the phenyl propanoic

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glucoside.

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Comparison between samples

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Initially the data was subjected to principle component analysis, but no clear groupings were

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observed, except for some outliers that had higher levels of PPAG (compound 29, sample 18 and

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19, Data in Table 3, Figure 9S, 10S, 11S).

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Upon closer inspection of the list of major compounds in the tea in Table 2, it is clear that there

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are groups of structurally related compounds (Figure 6, 7 and 8). The first group is related to the

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dihydrochalcone 3-hydroxyphloretin (produced from phloretin via chalcone-3-hydroxylase), that

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has been glycosylated via a C or O bond on different positions and with different sugars, of

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which glucose is the most abundant. The second group is related to phloretin dihydrochalcone

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and includes vitexin (compound 36), isovitexin (compound 39) and vicenin-2 (compound 22).

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Phloridzin (compound 51) is an O-glucoside and nothofagin (compound 45) a C-glucoside of

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phloretin.

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A probable pathway from 3-hydroxyphloretin via eriodictyol to quercetin (compound 56) is

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shown in Figure 8. This constitutes the third group of related compounds. In addition to

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quercetin itself, three different glycosylated quercetin derivatives were detected.

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The fourth group is related to coumaric acid (compound 28) and include E and Z

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phenylpropenoid acid glucoside (compounds 16,29, PPAG) and piscidic acid (compound 6). In

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the shikimic pathway, shikimic acid is converted to chorismate and the pathway is followed to

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either phenylalanine or tyrosine. Phenylalanine is converted to phenyl propenoic acid which is

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hydroxylated to hydroxy phenyl propenoic acid. We postulate that rooibos has an enzyme that

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can glycosylate hydroxy phenyl propenoic acid to form phenyl propenoic acid glucoside and its

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stereoisomer E-PPAG. When the concentrations (in mMol) of these four main groups were

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added up (after the concentrations were converted to mM), the relations between some of the

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samples from the same areas became clear. Figure 9 shows that there is still a large degree of

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variation in the different flavonoid profiles. The northern resprouters (samples 18 and 19, the

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Gifberg and Niewoudtville populations) showed much higher levels of PPAG when compared to

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plants from other areas. This possibly points to some sort of drought tolerance strategy

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developed by the plant to survive in the low rainfall (