Hydrolyzable Tannins, Flavonol Glycosides, and Phenolic Acids Show

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Hydrolyzable Tannins, Flavonol Glycosides and Phenolic Acids Show Seasonal and Ontogenic Variation in Geranium sylvaticum Anu Tuominen, and Juha-Pekka Salminen J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017

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Journal of Agricultural and Food Chemistry

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TITLE AND AUTHORSHIP

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HYDROLYZABLE TANNINS, FLAVONOL GLYCOSIDES AND PHENOLIC ACIDS SHOW

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SEASONAL AND ONTOGENIC VARIATION IN GERANIUM SYLVATICUM

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Tuominen, Anu* and Salminen, Juha-Pekka

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Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of

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Turku, FI-20500 Turku, Finland

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Corresponding Author

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*(A.T.) Phone +358 29 450 3207. Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The seasonal variation of polyphenols in the aboveground organs and roots of Geranium sylvaticum in

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four populations was studied using UPLC-DAD-ESI-QqQ-MS/MS. The content of the main

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compound, geraniin, was the highest (16% of dry weight) in the basal leaves after the flowering period

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but stayed rather constant throughout the growing season. The compound-specific mass spectrometric

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methods revealed the different seasonal patterns in minor polyphenols. The maximum content of

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galloylglucoses and flavonol glycosides were detected in the small leaves in May. Whereas the content

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of further modified ellagitannins, ascorgeraniin and chebulagic acid, increased during the growing

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season. In flower organs, the polyphenol contents differed significantly between ontogenic phases so

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that maximum amounts were typically found in the bud phase except in pistils the amount of

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gallotannins increased significantly in the fruit phase. These results can be used in evaluating the role

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of polyphenols in plant-herbivore interactions or in planning the best collection times of G. sylvaticum

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for compound isolation purposes.

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KEYWORDS: Geranium sylvaticum, wood cranesbill, seasonal variation, polyphenols, tannins,

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flavonoids, UPLC-DAD-MS, MRM quantification

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INTRODUCTION

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Geranium sylvaticum is a common herbaceous plant in Finland which grows wild in meadows, road-

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sides and herb-rich forests in the most parts of Europe. The extracts of other Geranium plants are

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widely used as traditional medicines against infectious and other human diseases because of their broad

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antiviral and antimicrobial activities.1-4 These bioactive properties are derived from the high content of

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hydrolyzable tannins; especially the main ellagitannin in Geraniums, geraniin, has been studied for its

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antioxidant activity, cancer prevention and hypoglycemic activity.4-5 Furthermore, tannin-rich plants or

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plant extracts have the potential to be used as feeds and feed additives for animals which reduce

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greenhouse gas emissions or possess antiparasitic properties.6-8 G. sylvaticum is a typical plant found in

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grasslands where ruminants feed and in in vitro screening assay it was a promising bioactive grass plant

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in reducing 30% methane and 80% ammonia emissions of ruminants and still having good organic

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matter digestibility.8

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The organs of G. sylvaticum contain a large variety of polyphenols, including ellagitannins,

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proanthocyanidins, gallotannins, galloyl (G) glucoses, galloyl quinic acids, flavonol glycosides and

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simple phenolic acids of which over 50 compounds were tentatively identified in our previous study

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using high-resolution mass spectrometry.9-11 There is intraplant variation in phenolic profiles between

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organs

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dehydrohexahydroxydiphenoyl (DHHDP)-ellagitannins, 9-10 that are rare in other Finnish plants.12 For

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the utilization of G. sylvaticum for medicinal purposes or as a feed additive, it is crucial to know if

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there is significant seasonal or ontogenic variation in the polyphenol contents of different plant organs.

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Such a variation might also have ecological implications as some of the tannins might function as

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defensive chemicals against herbivores and pathogens.

of

G.

sylvaticum

but

all

parts

contain

geraniin


and

other

geraniin-type


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The typical geraniin content (ca. 10% of DW) in Geraniums is so surprisingly high for a

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secondary metabolite it must have some special role for the plant.13 The seasonal variation in the

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geraniin and polyphenol content of G. sylvaticum has not been studied before, however, a few studies

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conducted with other Geranium species can be found. Results of these studies follow the overall

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patterns of herbaceous plants, in which the content of secondary metabolites increases during the

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ontogenic change from seedlings to mature plants.14 In G. lucidum leaves, the content of ellagitannins

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increased from 2.5 % (of fresh weight) in young seedlings to 4.5% (of fresh weight) in mature leaves.15

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Whereas in G. macrorrhizum, the content of hydrolyzable tannins was at the highest during flower

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budding state in the aboveground parts and during the seed formation in the rhizomes.2 Similarly in the

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fresh aboveground tissue of G. thunbergii, the content of geraniin was at the lowest in May (0.6 % of

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fresh weight) and at the highest during flowering in August (1.8 % of fresh weight) and then it

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decreased towards the end of the growing season in October (1.2 % of fresh weight).13 However, in the

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last two studies the all aboveground tissues were pooled together and therefore the compounds of

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flower stalks, flowers and leaves were mixed in the sample.

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The flowering of G. sylvaticum lasts three to four weeks from the end of May to mid-August in

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Finland depending on the altitude.16-18 Flowers usually grow in pairs in erect flower-stalks and on

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average each plant produce 12 flowers.19-20 The flowers of G. sylvaticum are heavily consumed by

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specialized herbivores.16 The flower petals are the main food source for the adults of oliphagous

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Zacladus geranii and they oviposit their eggs in the ovaries or in the style on the flowers.16, 21-23 After

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hatching, the larvae of the Z. geranii consume the developing seeds by either boring a small hole into

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the seed or eating a part of the it.16,

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Geranium flowers as herbivores and adult butterflies use the nectar of plants.24-25 The high herbivore

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pressure might be reflected to the contents of defensive compounds in the flower organs of G.

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sylvaticum during the ontogenic phases which makes it a particularly interesting study object.

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In addition, butterfly larvae and beetles can be found in the


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Our aim was to study the seasonal and ontogenic variation of tannins and other phenolic

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compounds in the leaves, flower parts and roots of G. sylvaticum. We also wanted to study separately

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the intraplant variation of phenolic compounds between the flowering shoot leaves and basal leaves.

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Furthermore, the difference between plant samples collected from four different populations in Turku

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were investigated. In particular, we address the following questions: 1) Does the content of

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hydrolyzable tannins, flavonoid glycosides and phenolic acids increase during temporal change from

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young tissues to mature ones as generally detected with herbaceous plants? 2) Does the seasonal trend

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of specific compound differ between plant organs? 3) Is the content of polyphenols higher in the

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flowering shoot than in the basal leaves due to the importance of flower shoot to the reproduction and

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the higher risk of herbivores that feed and pupate to the flower structures? 4) And does the content

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change when the seeds have been developed and spread? 5) Can the highest amount of defensive

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compounds be detected in flower parts during the most vulnerable bud phase? Our hypothesis is that

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by using more sensitive and selective mass spectrometric methods we could reveal seasonal variation

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trends in the content of minor compounds as well and be able to find answers to these questions.

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MATERIALS AND METHODS

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Chemicals and Reagents. Technical grade acetone (VWR, Haasrode, Belgium) was used

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for the extraction. Water was purified with a Millipore Synergy Water Purification system (Merck

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KGaA, Darmstadt, Germany). Chemicals used in the UPLC analysis were LC-MS grade acetonitrile

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(Sigma-Aldrich, Steinheim, Germany) and formic acid (VWR, Helsinki, Finland). Pure compounds

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used for the calibration curves were commercial or isolated from plant sources as listed in Tables 1 and

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2.



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Plant material. Plant samples of G. sylvaticum were collected from four different

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populations in Turku area. The voucher specimens (TUR 597241-597244) are deposited in the

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herbarium of the University of Turku. Study populations were: Oriketo 1 (N 60° 28.785; E 022°

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18.818) is a shadowed forest; Oriketo 2 (N 60° 28.832; E 022° 18.264) is a semi-shadowed forest;

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Oriketo 3 (N 60° 28.944; E 022° 18.096) is a sunny roadside population and Katariina (N 60° 24.752;

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E 022° 16.363) is a sunny meadow. Each population was divided into three smaller plots and every

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time plants were sampled from each plot.

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Three separate sample sets were collected: whole plants, leaves and flowers. First, a once a

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month sampling protocol was applied to whole plants: three plant individuals were collected from each

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population and stems, roots and leaves were separated. Secondly, to study seasonal variation in leaves

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more precisely, leaf samples from three different developing stages (small, medium, large) were

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collected altogether 16 times; five times in 5-day intervals in May and June and three times in July,

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twice in August and once in September. Four leaves from each three plots of population were pooled

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together. Thirdly, during the main flowering period in July also flowers were collected to study their

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ontogenic variation. Flower organs were carefully separated by hand and samples of three flowers from

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the same individual were combined to achieve enough sample material for analysis.

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Sample preparation and Extraction. All samples were freeze-dried and homogenized

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into a fine powder using ball grinder. The whole sample of small organs (0.5–30 mg) and 10 mg aliquot

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of larger samples, such as roots and leaves, were used for the 4x30 min extraction with 700 µL

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acetone:water (7:3, v/v) using vortex. Samples were centrifuged for 10 min and supernatants were

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combined to another Eppendorf tube for the evaporation of acetone in an Eppendorf concentrator. The

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water-phases were lyophilized, re-dissolved and diluted to varying volumes of ultrapure water


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depending on their initial weight and filtered through 0.2 µm PTFE syringe filters before UPLC

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analysis.

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UPLC-MS/MS Analysis. The quantitative analyses were performed with an ultra-high

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performance liquid chromatographic system (UHPLC, Acquity UPLC®, Waters Corporation, Milford,

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MA, USA) combined with a triple quadrupole mass spectrometer (Xevo® TQ, Waters Corporation,

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Milford, MA, USA) and a diode array detector. An Acquity UPLC® BEH Phenyl (2.1 × 100 mm i.d.,

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1.7 µm, Waters Corporation, Wexford, Ireland) column was used. Column oven temperature was 40

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°C. The UV spectra were recorded between 190–500 nm. The LC flow rate was 0.5 mL/min. The

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gradient profile of eluents acetonitrile (A) and 0.1 % formic acid (B) was as follows: 0–0.5 min, 0.1%

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A in B; 0.5–5.0 min, 0.1–30.0% A in B (linear gradient); 5.0–8.0 min, 30.0–45.0% A in B (linear

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gradient); 8.0–8.1 min, 45.0–90.0% A in B (linear gradient); and 8.1–11.5 min, column wash and

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stabilization. The injection volume was 5 µl. The mass spectrometer was operated in the negative ESI

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mode using following general parameters: The capillary voltage was set at 3.4 kV, the source

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temperature at 150 °C, the desolvation temperature at 650 °C, the desolvation gas (N2) flow was set at

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1000 l/h and cone gas (N2) flow 100 l/h. Collision gas was argon. The compound relating parameters of

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multiple reaction monitoring (MRM) methods were optimized using plant fractions or isolated

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compounds (Tables 1 and 2). Different set of MRM methods were used to each organ samples based on

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what were the most prevalent compounds in that organ. The amount of proanthocyanidins, PC% and

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mean degree of polymerization (mDP) was measured from roots with methods previously published.28

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The performance of MS/MS system was controlled using a mixture of 4 µg/mL flavonoids

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(kaempferol-7-O-glucoside, kaempferol-7-O-neohesperoside, kaempferol-3-O-glucoside, quercetin-3-

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O-galactoside, and quercetin 3-O-glucoside) at the beginning of the batch and 1 µg/mL catechin (both

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prepared in 1:4 acetonitrile/0.1% formic acid, v/v) solution that was injected five times after every ten



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samples. Calibration curves were prepared for compounds, that were available as pure compounds from

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previous studies, and others were quantified as equivalents of the closest similar compound (Tables 1

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and 2).

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Statistical Analysis. The difference between different sized plant parts, months and

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populations were calculated using t-test, mixed model, one-way ANOVA and Welch’s ANOVA, when

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the homogeneity of variances was not met in all groups. Tukey’s standardized grouping was used to

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differentiate the months. All statistical analyses were performed using SAS add-on in Excel.

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RESULTS AND DISCUSSION

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The phenolic profiles of G. sylvaticum organs are complex because extracts contain hundreds of

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compounds.9-10 Figure 1 shows overview of G. sylvaticum plant, its organs and the main polyphenols in

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each organ. Schemes 1–3 present examples of polyphenol structures which we have identified earlier

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from G. sylvaticum using high resolution mass spectrometry, and which seasonal variation was now

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studied.

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Intraplant variation of polyphenols in leaves. The first leaf sample set was collected

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to study the intraplant variation between two different types of leaves: larger and long-stalked basal

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rosette leaves and stem leaves, which contained both short-stalked leaves and the smaller stipulate

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leaves of the flowering shoot (Fig. 1). Each G. sylvaticum plant has one to several flowering shoots and

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several basal leaves.20 It was found that the content of geraniin in the stem leaves (153 ± 28 mg/g, n =

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44) and basal leaves (153 ± 23 mg/g, n = 55) did not differ when all populations and months were

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calculated together. There was not statistically significant difference between basal and stem leaves in

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the other polyphenols contents either (Table S1). The mixed ANOVA calculations showed that part


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alone is not a significant source of variation for polyphenols contents in G. sylvaticum leaves although

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when part and month or part and population together can have significant effects (Table S1). For

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example, when geraniin contents were calculated per month, it was revealed that the seasonal pattern

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for varied between the basal and stem leaves (Fig. S1). The difference was the clearest at the prime

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time of flowering in June, when the content of geraniin was 160 ± 19 mg/g in the stem leaves and 138

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± 29 mg/g in the basal leaves (Fig. S1). It seemed that during the blooming more geraniin was allocated

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to the flower stalk, but after seeds were ripe and spread and the flower stalk started to decompose, the

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amount of geraniin declined. Whereas in the basal leaves, the content of geraniin increased after

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flowering because the plant did not need to invest in the flower shoot no longer (Fig. S1). This pattern

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was not equally clear for carpinusin, an isomeric ellagitannin for geraniin, as its amount stayed more

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stable. Furthermore, ascorgeraniin and chebulagic acid followed a different pattern: contents increased

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significantly during the summer in the both flower stalks and basal leaves (Fig. S1).

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Variation in polyphenols contents between populations in leaves. The leaf

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samples were collected from four different populations in Turku area to compare the variation in

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polyphenol contents caused by differences in the growing conditions and locations. The Katariina

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population is situated far away from the other three populations; it was a sunny meadowside next to a

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forest and a nature conservation area. This meadow was cut down in July. Three other populations are

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situated more closely to each other and can therefore be expected to be more similar to each other.

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However, the main flowering period and seed maturing times differed slightly between populations so

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that the high-light populations Katariina and Oriketo 3 had longer flowering periods.

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The mixed ANOVA calculations showed that population is meaningful source of variation for

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polyphenols contents in G. sylvaticum leaves (Table S1). The results of geraniin contents in leaves

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demonstrate how the contents and seasonal pattern were different between four studied populations



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(Fig. 2). The content of geraniin was the highest in Oriketo 3 population during the whole summer.

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Whereas, in Oriketo 2 population, the content of geraniin was clearly the highest in the spring and after

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that decreased to lower level than in other populations (Fig. 2). In addition, the overall contents of

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flavonoids, tetraGG and galloyl quinic acids were the highest in the Oriketo 3 population (data not

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shown). Also Katariina population, which located in a sunny field, exhibited the significantly higher

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contents of many ellagitannins and quercetin mono- and diglycosides than Oriketo 1 and 2 populations

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which have more shadowed forest conditions. Exceptions were the content of carpinusin and galloyl

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shikimic acids which were clearly higher in Katariina and Oriketo 1 populations than in the other

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populations (data not shown). Overall, it seemed that the stressful conditions, for example high-light

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and dryness, causes the polyphenol content to stay higher in Oriketo 3 and Katariina populations than

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in other Oriketo populations although the location is closer to each other.

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Seasonal variation of polyphenols in leaves. The seasonal variation of different

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polyphenols was thoroughly investigated especially with leaf samples because leaves are the most

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potential part for harvesting among the plant organs of G. sylvaticum due to its high ellagitannin

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content. The leaves in the second sample set were collected more frequently and sample set tested also

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the variation between leaf size in basal leaves. It was possible to collect leaves of three size in all

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sampling dates because new leaves are continuously formed throughout the growing season. This way

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it was possible to see, if the phenolic profile is more dependent on the size of the plant than the date of

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the collection. The average of leaf dry weight was many times lighter among small sized leaves (33 ±

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12 mg, n = 64), than among medium sized (116 ± 31 mg, n = 64) and large sized (262 ± 69 mg, n = 64)

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leaves. The results in tables 3-9 are expressed as concentrations of polyphenols, which are relevant

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when we consider leaf as a material or the plant quality to herbivores. However, to see how the


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dilution, when leaf grows from small to large, changes the observed seasonal trends, we calculated

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selected results also as quantity per leaf taking the leaf mass account (Figs. S2-S7).

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The content of the main compound, geraniin, was rather constant throughout the sampling

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period (Figs. 2 and 3, and Table 3). The highest contents of geraniin were detected in July after the

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flowering period had ended and the seeds were in the maturing phase. Small-sized leaves had

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significantly higher contents of geraniin (157 ± 19 mg/g DW, n = 64) in comparison to medium and

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large leaves during the summer in all populations (ANOVA, p=0.0000) (Fig.2). Geraniin content in

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medium (147 ± 19 mg/g DW, n = 64) and large (142 ± 18 mg/g DW, n = 64) sized leaves did not differ

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significantly.

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However, significant seasonal variation was detected in the contents of minor hydrolyzable

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tannins. In the present study, the use of compound-specific MRM methods enabled the selective

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detection of minor ellagitannins of which reliable quantification is difficult using an UV detection

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because of the co-eluting compounds. Figure 3 shows that there was three different kinds of seasonal

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trends in the contents of selected hydrolyzable tannins when amounts of all sized leaves in all

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populations were calculated together. The seasonal pattern of carpinusin was similar to that observed

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with geraniin; the content remained rather constant during summer and the maximum content was

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found in the small leaves in August (50 ± 25 mg/g DW) (Fig. 3 and Table 3). However, the difference

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between the leaves of different size was not very profound. A similar pattern was observed for the

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concentrations of G-hexahydroxydiphenoyl (HHDP)-glucose B, digalloyl-HHDP-glucose and

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geraniinic acid (Table 3 and Fig. 3). In contrast, the content of ascorgeraniin and chebulagic acid,

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which are further modified types of geraniin (Scheme 2), increased more than three times towards the

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end of the growing season when compared with their content in May (Table 3 and Fig. 3). Their

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content was higher especially in large sized leaves (Figs. S2-3).



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Whereas, the greatest amount of trigalloyl-HHDP-glucose was found in May and in small sized

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leaves (Fig. 3). The content of galloylglucoses followed a clear seasonal trend which was similar to that

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of trigalloyl-HHDP-glucose. The amount of galloylglucoses was many times higher in the small leaves

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of all populations collected in May than in the larger leaves collected in June when the flowering

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period had begun (Table 3 and Fig. 3). This pattern was clearest in the small leaves and galloylglucose

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content stayed higher for longer time period, but it was also visible in the medium and large sized

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leaves in May. After May, the content of galloylglucoses remained rather low (ca. 5 mg/g) and constant

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in all sized leaves leaves in all populations. The difference between sampling dates was statistically

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significant also when calculated as per leaf mass in the small and medium sized leaves for pentaGG

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and monoGG (Figs. S4-6). Thus, this pattern was more related to the month than the leaf size. PentaGG

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(Scheme 1) is a precursor for triG-HHDP-glucose and tellimagrandin II, and these simple ellagitannins

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can be further modified to ellagitannins containing DHHDP groups (Scheme2). Galloylglucoses are

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known precursors to other more complex hydrolyzable tannins and a similar seasonal trend for

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galloylglucoses has been observed in the studies of oak and birch leaves.31-34

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The seasonal trend of galloyl shikimic acid resembled that of galloylglucoses and the difference

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in the small leaves was statistically significant also when the leaf size was taken account (Figs. 4 and

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S8). Whereas the content of monoG quinic acids, brevifolin carboxylic acid and chlorogenic acid

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stayed more constant during summer (Fig. 4). The seasonal patterns of flavonol glycosides varied

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depending on the aglycone skeleton (Fig. 4 and Table 3). The content of quercetin derivatives in leaves

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was the highest in spring and decreased to half towards the fall (Fig. 4). The amounts of quercetin

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galloylglycoside were slightly higher in large than in small sized leaves but difference was no

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statistically significant between difference sized leaves (Fig. S9). In contrast, the amount of kaempferol

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derivatives stayed more constant throughout the season (Fig. 4, Table 3).


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Generally it is thought that herbs and woody plants have different temporal patterns in their

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contents of secondary metabolites because of the different life spans: The amount of metabolites

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increases with ontogeny in herbs that live only from one to few years whereas woody plants which can

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live tens of years show also seasonal changes.14 HPLC studies of individual polyphenols with tree

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leaves have showed that the different polyphenol groups can exhibit different seasonal trends. The

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content of galloylglucoses, ellagitannins, p-coumaroyl quinic acid derivatives and flavonoid glycosides

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in oak and birch leaves is at the highest in early spring and decreases towards the end of the growing

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season, whereas the content of proanthocyanidins is the highest in the mature leaves.31-34 In addition,

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the structures of tree leaf hydrolyzable tannins have been observed to change during the growing

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season. These changes follow the biogenetic pathway so that the simple galloylglucose precursors

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detected in early spring transform to more complex structures that contain more galloyl and oxidatively

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coupled HHDP groups during the growing season (Schemes 1–2).31-32, 35 In contrast to woody plants,

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Okuda and Ito (2011) have stated about hydrolyzable tannin structures in herbaceous plants that these

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stay basically constant until the leaves decay.36 However, the present results evidenced that

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hydrolyzable tannin structures in herbaceous plants can also be transformed during the growing season:

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The contents of galloylglucoses and phenolic acids, that are precursors to more complex hydrolyzable

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tannins, were higher in spring, and the amount of further modified ellagitannins increased towards the

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end of the growing season.

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Seasonal and intraplant variation of polyphenols in stems. The stems and leaves

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of G. sylvaticum have similar polyphenol profile.9 The amount of geraniin in the stems was much lower

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than in the leaves and varied between 5 and 57 mg/g of DW (Table 4). Similarly to leaves, the highest

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amount of geraniin in stems was detected in the Oriketo 3 population (data not shown). Other major

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ellagitannins in stems were G-HHDP-glucose B and carpinusin (Table 4). The ratios of G-HHDP-



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glucose B:geraniin 1:3 and carpinusin:geraniin 1:1.6 were high in comparison with the corresponding

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ratios detected in leaves, 1:8 and 1:5 respectively. There was no significant difference between the

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polyphenol content of leaf stalks (n= 53) and flowering shoots (n=47) when all populations and

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sampling months were pooled together (data not shown). However, seasonal differences were detected.

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In basal leaf stalks the amounts of ellagitannins and galloyl quinic acids were lower in the spring and

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increased towards the maximum content in August, when flowering period had ended (Table 4).

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Whereas, the polyphenol content in flowering shoots was more uniform throughout the growing season

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and started to decrease slowly when the seeds were spread and stems begin to turn brown and dry.

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The amounts of GGs, gallotannins and proanthocyanidins were very low in stems. The content

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of monoGG was the highest in May, similarly to leaves (Table 4). Instead of typical GGs, two new

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compounds were detected in stems, which were tentatively identified to be methylated mono- and

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digalloylglucoses using high-resolution MS (data not shown). They were not present in leaves, but

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small amounts were detected in the stamens and pistils as well. The presence of methylated monoGG

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has been found previously in the related Erodium species.37 Methylated GGs were most abundant in

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Oriketo 1 population and their content increased towards the fall although the difference was not

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statistically significant (Table 4). Whereas Katariina population was clearly different with significantly

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less amounts of these methylated compounds (data not shown). The amount of flavonoids was

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relatively constant in stems, a slightly higher concentration of quercetin glycosides was observed in

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spring and their amount decreased towards the fall as observed with leaves. There was no significant

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difference in the flavonoid contents between leaf stalks and flower shoots (Table 4).

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Seasonal variation of polyphenols in roots. The high content of proanthocyanidins in

310

G. sylvaticum roots causes a hump to the UV chromatogram of root extract, which complicates the UV


Page 15 of 49


15 311

detection of compounds that elute at the same time such as geraniin and other ellagitannins.9

312

Furthermore, the similarity of the UV spectra of phenolic compounds prevents the use of extracted UV

313

chromatograms for quantification. Therefore MRM methods, which utilize the mass transitions, were

314

especially useful for the selective detection of compounds from the G. sylvaticum root extracts more

315

reliably.

316

Polyphenols of the root samples did not exhibit statistically significant seasonal variation during

317

the summer except that the content of diG-HHDP-glucose (31 ± 23 mg/g DW) was the highest in

318

August (Table 5). The amount of carpinusin was almost equal to geraniin (38 ± 18 mg/g DW) (Table

319

5). In addition, the amounts of other hydrolyzable tannins were at maximum in July or in August

320

although differences were not statistically different (Table 5). This seasonal trend resembles the results

321

observed with G. macrorrhizum, in which the content of hydrolyzable tannins was at the highest during

322

the seed formation in the rhizomes.2 Roots contained a relatively high amount of galloyl quinic acids.

323

The amount of monogalloyl quinic acids was the highest in July (Table 5). Furthermore, the

324

proanthocyanidin content showed seasonal variation and it increased from June to August significantly.

325

However, there was no statistically significant seasonal change in the mean degree of polymerization

326

(mDP) of proanthocyanidins or in the PC/PD ratio (Table 5).

327

There was some variation in the polyphenol contents of roots between populations (Fig. 5).

328

Oriketo 2 differed from other populations with the significantly lower content of geraniin and

329

carpinusin and other ellagitannins (Fig. 5) as observed also in leaves (Fig. 2). Furthermore, Katariina

330

population contained more procyanidins than the other populations whereas the total content of

331

proanthocyanidins did not differ between populations (Fig. 5).

332

Ontogenic variation in flower parts. At first, when the flower of G. sylvaticum opens, is

333

the male phase where the anthers of stamens present pollen (Fig. 6). The lobes of the pistil stigma



Page 16 of 49

16 334

remain closed during that phase to prevent self-pollination. When the anthers are dropped, starts the

335

female phase where stigma lobes are open and receptive to pollen (Fig. 6).38 After pollination stigma

336

lobes close again and petals drop.17, 38 G. sylvaticum flowers remain open only from 3 to 80 hours;

337

duration depends on the weather conditions, insect visitation rates and about the sexual morph.17, 38 So

338

instead of seasonal variation, ontogenic changes in different life phases of the flowers of G. sylvaticum

339

were studied and samples were divided into three groups based on these flower phases (Fig. 6). Results

340

from all populations were calculated together in Tables 6–9 due to the small number of samples.

341

Ontogenic variation of polyphenols in sepals. One G. sylvaticum flower bud has five sepals.

342

Many flower buds suffer from herbivore damage before the flowering and often there are only small

343

holes in the sepals but the all other flower parts inside the sepals have been eaten (personal

344

observation). After the flowering, sepals turn brown and dry (Fig. 6). Sepal samples were divided into

345

three size groups. The smallest leaves were collected from buds and medium and large sized sepals

346

from the open flowers. The weight of the sepals only slightly increased from small to large sepals

347

(Table 6). Sepals differed from other plant organs of G. sylvaticum so that the amount of carpinusin in

348

the G. sylvaticum sepals is notably high, equal to the content of geraniin in the leaves (Table 3). The

349

amount of carpinusin was the highest in the small leaves (160 ± 59 mg/g DW) and decreased in the

350

larger leaves (133 ± 57 mg/g DW) but the difference was not statistically significant (Table 6).

351

There was no significant ontogenic variation in polyphenol content of sepals (Table 6). Sepals

352

contained only small amounts of other galloylglucoses and gallotannins (less than 2 mg/g DW) and

353

therefore, their contents are not presented in Table 6. However, the content of other GGs was higher in

354

small sepals as observed in the leaves also. The dominating flavonoids in the sepals were quercetin

355

galloylglycosides and other quercetin derivatives, the same as observed in the leaves (Table 6 and 3).

356

The content of flavonoids was somewhat higher in the small sized leaves. The amount of quinic acids

357

stayed similarly constant during the growing season (Table 6). The G. sylvaticum flower buds can be


Page 17 of 49


17 358

formed already in the year before flowering and enclosed in the winter bud.18 Therefore, the used

359

sample collection did not really cover the true ontogenic change in the sepals of G. sylvaticum.

360

Ontogenic variation of polyphenols in petals. We stated earlier that because of the short

361

lifespan of G. sylvaticum flowers, the plant does not seem to invest much defensive compounds in

362

petals, instead it synthesizes more pigments, i.e. compounds that invite insects such as pollinators.9-11

363

The petals of G. sylvaticum contain sylvatiins, rare acetylglucosylated hydrolyzable tannins that can act

364

as co-pigments of anthocyanins enhancing their color (Scheme 3).11 Our hypothesis was that the

365

content of sylvatiins might be the highest during the open phase of flowers when the plant tempts

366

pollinators. However, the amounts of the main sylvatiins C and D stayed rather constant in all

367

ontogenic phases as did the amount of other pigments acetylmalvin and the main flavonoids (Table 7).

368

In contrast, the amounts of galloylglucoses and corresponding small sized sylvatiins were significantly

369

higher in the smaller petals than in fully open ones which can indicate that these compounds are

370

precursors to other more complex sylvatiins and hydrolyzable tannins (Scheme 3). The proposed

371

structural relation of sylvatiins (Scheme 3) follows the biosynthetic routes of galloylglucoses and

372

hydrolyzable tannins (Schemes 1–2), so that for example, sylvatiin A that has pentagalloylglucose core

373

with one acetylglucose attached, is precursor for sylvatiin D that has chebulinic acid core, and for

374

sylvatiin C that has two acetylglucose parts. The contents of geraniin (from 41 mg/g to 10 mg/g),

375

carpinusin (from 24 mg/g to 4 mg/g) and other ellagitannins were higher in small sized petals inside the

376

buds than in the fully open flowers (Table 7). This difference was statistically significant (Table 7).

377

The size of a petal increases notably between phases (Fig. 6 and Table 7) and therefore the

378

differences between phases were calculated also as polyphenol amounts per petal unit (Fig. S7). When

379

calculated this way, the amounts of most of the studied polyphenols were higher in open flower phase

380

than in the bud phase (Fig. S7). For example, the amounts of the main anthocyanin, acetylmalvin, and



Page 18 of 49

18 381

its co-pigments sylvatiin D and kaempferol diglycosides were statistically significantly higher in open

382

flowers and during pollination as hypothesized (Fig. S7).

383

Ontogenic variation of polyphenols in stamens. G. sylvaticum has ten stamens that are in two

384

whorls of five, the filaments of inner whorl can be slightly longer and its anthers earlier in the blue

385

mature stage.26, 38 The stamen samples were divided into three groups based on the ontogenic phases

386

for the statistical analysis. The first group contained mainly stamens from the bud phase when anthers

387

are still immature and yellow. The second group contained samples of the pollen presentation phase,

388

and the third one contained filaments after the anthers have fallen off (Fig. 6). The weight of one

389

stamen varied between 42 and 476 µg, there was a huge decrease in weight when the anthers had fallen

390

off after the pollen presentation phase (Table 8).

391

The content of hydrolyzable tannins was slightly higher in the bud phase and in the filaments

392

than in the stamens during the open phase when the pollen is mature. The difference was the largest for

393

geraniin which content in stamens during the bud phase was 33 ± 12 mg/g and in the open flower phase

394

19± 9 mg/g (Table 8). The content of geraniin in filaments was 28 ± 16 mg/g which indicated that

395

geraniin is mainly situated in them. Carpinusin had a similar trend although its content was one third of

396

the content of geraniin (Table 8). Interestingly, the contents of pentaGG or other GGs did not show

397

ontogenic trends in the stamens which indicated that the further galloylation was not related to the

398

maturation of stamens. Also, traces of gallotannins were detected in stamens with MRM methods

399

which content did not change significantly during the different phases (Table 8).

400

Stamens contained notably high content of chebulagic acid, when compared with other G.

401

sylvaticum organs. Chebulagic acid was an exception among the hydrolyzable tannin group in stamens

402

so that its content was the highest in the mature pollen presentation phase (Table 8). After the fall out

403

of purple-coloured anthers, the contents of chebulagic acid, malvin, and kaempferol diglycoside A


Page 19 of 49


19 404

significantly decreased, which indicated that these compounds were mainly situated in the anthers and

405

pollen (Table 8). Especially, the content of kaempferol diglycoside A was very high in the bud phase,

406

decreased notably after the pollen presentation and almost nothing were detected in the filaments. This

407

might indicate that this flavonoid is a precursor for anthocyanins or a co-pigment, which are needed

408

during the pollen presentation phase, when the color of anthers turn blue as a signal to the pollinators

409

that the pollen is mature and ready for the pick-up. This fits to the hypothesis that kaempferol

410

glycosides might have a special role in the stamens related to pollen germination.9-10 Other flavonoids,

411

also the isomeric kaempferol diglycoside B, showed an opposite seasonal pattern in which there were

412

less flavonoids in the stamens during bud phase than in the open flowers (Table 8).

413

Ontogenic variation of polyphenols in pistils. G. sylvaticum pistils have five stigmatic lobes

414

that are closely joined to each other in a male phase, and then curve out and unfold when the female

415

phase starts and stigmas are receptive to pollen (Fig. 6). After pollination, the fruit stage begins when

416

the ovary chambers become clearly bigger and style longer.23, 39 The fruit changes from green to brown

417

just before the seeds are mature for ejection, about three weeks after flowering (Fig. 6).18, 24 The pistil

418

samples of G. sylvaticum were divided into three groups based on their sizes. In the group of small

419

sized pistils, the pistils were from buds or just opened flowers that were in the male phase presenting

420

pollen. In medium sized pistil group, most of the pistils were in the female phase. The third group

421

contained pistils in the fruit phase where the seeds were already developing inside the ovary (Fig. 6).

422

After the pollination, the pistil weight started to grow and the weight was ten times higher in the fruit

423

phase (Table 9).

424

The hydrolyzable tannin profile of G. sylvaticum pistils was quite similar to leaves and sepals

425

except that the pistils contain a distinctive amount of gallotannins.9-10 Interestingly, the amount of

426

geraniin decreased when the fruit developed whereas the content of carpinusin and diG-HHDP-glucose

427

increased (Table 9). The amount of galloyl quinic acids stayed relatively constant in all sized pistils



Page 20 of 49

20 428

(Table 9). Two marker compounds, which are typical for G. sylvaticum seeds,9 reflected the presence

429

of developing seeds inside the ovaries; the amount of tryptophan and catechin increased in the fruit

430

phase. Also, the amount of galloylated quercetin flavonoids increased towards the fruit phase whereas

431

the total amount of kaempferol glycosides was at the highest in the female phase (Table 9).

432

The most interesting compounds in pistils were gallotannins. The di-, tri and pentaGG

433

(1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose) contents decreased when the amounts of tetraGG,

434

heksaGG and heptaGG increased heavily during the ontogenic changes (Table 9). In addition to

435

1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose, a separate broad peak was detected, which exhibited

436

ions at m/z 939, 787 and 393, similar to that of pentaGG. However, the MRM method used for

437

pentaGG did not give a good response to that peak. A separated MRM method was optimized for it

438

which showed that the loss of galloyl unit (-152 Da) was more prominent to this compound than to

439

1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose. This is a typical fragmentation pattern for metadepsidic

440

galloyl groups found in gallotannins and therefore, it was concluded, that this peak contained isomers

441

of pentaGG, which contain galloyl groups attached with depside bonds. Similar pentaGG isomers

442

having two different cores 1,2,3,6-tetraGG with depsides at C-2 and C-6 and 1,2,3,4,6-pentaGG core

443

with depsides at C-2,-3, -4, -6-positions have been found in Turkish galls that contain also substantial

444

amount of gallotannins.40 Similarly to other gallotannins, the broad peak shape indicated that several

445

isomers were present. The amount of metadepsidic pentaGG was highest in the fruit phase (Table 9).

446

Thus a clear trend towards more galloylated hydrolyzable tannins during the active growth phase of

447

fruit was observed in the pistils of G. sylvaticum. Pistils were an exception, because the content of

448

polyphenols mainly decreased from the bud phase to the mature phase in the other flower organs of G.

449

sylvaticum (Fig. 6).

450


Page 21 of 49


21 451

To conclude, by using selective MRM methods it was possible to reveal that the different

452

organs show different seasonal patterns or ontogenic variation. Furthermore, the compound-specific

453

methods were able to reveal differences in the seasonal patterns inside a certain polyphenol group.

454

Typically all the aboveground tissues are pooled together in plant studies and therefore contain also the

455

compounds of flower stalks and flowers in addition to leaf compounds. Our study showed that there

456

can be delicate intraplant differences in seasonal trends in phenolic contents and the changes due to

457

ontogenic phases in flowers yielded totally different patterns for phenolic contents. It was observed that

458

G. sylvaticum contained a high content of many potentially bioactive compounds that can be isolated

459

for different purposes. The content of many polyphenols exceeded the level of 5 % of plant DW and

460

can then be considered to be meaningful as quantitative defensive compounds against herbivores. The

461

content of the main compounds in each organ stayed rather constant during the growing season; more

462

variation was detected in the amount of minor compounds such as galloylglucoses which content was

463

clearly higher in the spring and in the young tissues. Although these trends might be more related to the

464

biosynthetic pathways presented in the Schemes1-3 than the antiherbivore actions.

465

However, the presented results should be seen as relative concentrations, which can be

466

compared against each other inside the study, and not an absolute ones. For example, the ionization of

467

geraniin was enhanced due to the matric effects in the extract when compared to the pure compound

468

used for calibration curves (data not shown). In addition, many of the presented concentrations were

469

expressed as equivalents, which can also overestimate the concentrations.

470

To yield the highest amount of polyphenols, the best places to collect G. sylvaticum plants are

471

sunny and open populations in the yields or road sides. The amount of geraniin is highest in the small

472

basal leaves after the flowering was ended in August. The highest amount of galloylated flavonoids and

473

galloyl shikimic acids can be achieved from basal leaves in May and early June. The optimal time to

474

collect petals with rare sylvatiins and other hydrolyzable tannins is in the bud phase. These results



Page 22 of 49

22 475

show that the sepals are an especially good source of carpinusin because there is low amount of

476

interfering compounds, such as flavonoids present, and the content of carpinusin stays high in all

477

phases. The G. sylvaticum pistils are a good source of geraniin and gallotannins. To achieve maximum

478

yield in the isolation of gallotannins, pistils should be collected in the fruit phase before the seeds are

479

mature. Roots are the best source of proanthocyanidins and galloyl quinic acids as their content stayed

480

rather constant throughout the summer. This is the first survey were the seasonal variation of

481

polyphenol content of G. sylvaticum organs was studied in detail. These results can be used in

482

evaluating the role of different polyphenol groups as defensive compounds and seasonal patterns in the

483

related geranium species and other herbaceous plants as well.

484 485

ABBREVIATIONS USED

486

DAD, diode array detector; UHPLC, ultrahigh-performance liquid chromatography; QqQ, triple

487

quadrupole; MS, mass spectrometry; MRM, multiple reaction monitoring; ESI, electrospray ionization;

488

G, galloyl group; HHDP, hexahydroxydiphenyl; DHHDP, dehydrohexahydroxydiphenoyl; DW, dry

489

weight; GG, galloylglucose, ET, ellagitannin, PA, proanthocyanidins; HT, hydrolyzable tannin; mDP,

490

mean degree of polymerization.

491 492

ACKNOWLEDGMENTS

493

Authors want to thank Salla Timonen and Matias Niemelä for their help in the sample preparation, and

494

Anne Koivuniemi, Jussi Suvanto and Marica Engström for their help in UPLC-MS/MS quantifications.

495

Maarit Karonen helped to improve the earlier version of the manuscript.

496 497

FUNDING SOURCES


Page 23 of 49


23 498

This work was funded by grants from the Emil Aaltonen Foundation and Turku University Foundation

499

(to A.T.). The chemical analyses using the UPLC-MS system were made possible by a Strategic

500

Research Grant of the University of Turku (Ecological Interactions).

501 502

SUPPORTING INFORMATION DESCRIPTION

503

Supplementary information includes statistical data for the ANOVA calculation of polyphenols in G.

504

sylvaticum leaves, the figure of intraplant variation of selected ellagitannins in G. sylvaticum leaves and

505

the seasonal trends of selected compounds calculated as amount per organ unit in the leaves and petals.



Page 24 of 49

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(38) Varga, S.; Nuortila, C.; Kytöviita, M.-M. Nectar sugar production across floral phases in the gynodioecious protandrous plant Geranium sylvaticum. PLOS one 2013, 8:e62575-e62575, 1–6. (39) Varga, S.; Kytöviita, M.-M.; Siikamäki, P. Sexual differences in response to simulated herbivory in the gynodioecious herb Geranium sylvaticum. Plant Ecol. 2009, 202, 325–336. (40) Nishikawa, M.; Yamagishi, T. Tannins and related compounds. Part 9. Isolation and

603

characterization of polygalloylglucoses from Turkish galls (Quercus infectoria). J. Chem. Soc.

604

Perkin Trans. I, 1983, 961–965.

605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621


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29 622

FIGURE CAPTIONS

623 624

Figure 1. The plant organs of G. sylvaticum that were studied and the main polyphenols in each organ.

625 626

Scheme 1. Example structures of phenolic acids, proanthocyanidins, flavonol glycosides,

627

galloylglucoses and gallotannins, and the simplified biosynthetic route of galloylglucoses to

628

gallotannins.

629 630

Scheme 2. Structures of ellagitannins found in G. sylvaticum and their proposed structural relations.

631 632

Scheme 3. Structures and the proposed biosynthetic route of sylvatiins, and the main anthocyanin

633

found in the petals of G. sylvaticum.

634 635

Figure 2. Seasonal variation in the content of geraniin in small (black dotted line), medium (grey − −

636

−), and large (black line) leaves in A) Katariina, B) Oriketo 1, C) Oriketo 2 and D) Oriketo 3

637

population and their main flowering and seed dispersal periods.

638 639

Figure 3. The seasonal variation of individual hydrolyzable tannins in G. sylvaticum leaves from four

640

populations: Contents of ellagitannins are averages of all sized leaves in all four populations with

641

standard deviation (G, galloyl; HHDP, hexahydroxydiphenoyl).

642 643

Figure 4. The seasonal variation of some flavonol glycosides and phenolic acids in G. sylvaticum

644

leaves from four populations: Contents are averages of all sized leaves in all four populations with

645

standard deviation.



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30 646 647

Figure 5. Variation in contents of A) geraniin, B) carpinusin, C) PA total and D) PC% between

648

populations in roots of G. sylvaticum.

649 650

Figure 6. The ontogenic phases of G. sylvaticum flowers and the simplified ontogenic trend of

651

polyphenols.

652 653 654


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31 FIGURE GRAPHICS

Figure 1. The plant organs of G. sylvaticum that were studied and the main polyphenols in each organ.



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32

Figure 2. Seasonal variation in the content of geraniin in small (black dotted line), medium (grey − − −), and large (black line) leaves in A) Katariina, B) Oriketo 1, C) Oriketo 2 and D) Oriketo 3 population and their main flowering and seed dispersal periods.


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33 90

5.0

MonoG glucose

80

DW content mg/g

40 30 20

DW content mg/g

50

3.5 3.0 2.5 2.0 1.5

5.0 4.0 3.0 2.0

1.0

10

0.5

1.0

0

0.0

0.0

200

80

Geraniin

180

3.5

Carpinusin

70

DW content mg/g

160 140 120 100 80 60 40

60 50 40 30 20

2.5 2.0 1.5 1.0

20

10

0.5

0

0

0.0

8.0

25

Ascorgeraniin

40

Chebulagic acid

7.0

Date

G-HHDP-glucose B

35

6.0 5.0 4.0 3.0 2.0

DW content mg/g

20

DW conent mg/g

DW content mg/g

DiG-HHDP-glucose

3.0

DW content mg/g

DW content mg/g

60

TriG-HHDP-glucose

6.0

4.0

70

DW content mg/g

7.0

PentaG glucose

4.5

15

10

5

30 25 20 15 10 5

1.0

0

0

0.0

Date

Date

Date

Figure 3. The seasonal variation of individual hydrolyzable tannins in G. sylvaticum leaves from four populations: Contents of ellagitannins are averages of all sized leaves in all four populations with standard deviation (G, galloyl; HHDP, hexahydroxydiphenoyl).



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34

70

9.0

Quercetin galloylglycoside

7.0

Quercetin glycoside

Quercetin diglycoside

8.0

60

6.0

40 30 20

DW content mg/g

DW content mg/g

DW content mg/g

7.0 50

6.0 5.0 4.0 3.0

5.0 4.0 3.0 2.0

2.0 10 0

1.0

1.0

0.0

0.0

30

35

2.5

30

20 15 10

1.5

1.0

25 20 15 10

0.5

5

5

0

0

0.0

18

10

30

Monogalloyl quinic acid

Monogalloyl shikimic acid

9

25

12 10 8 6

20 15 10

4

7 6 5 4 3 2

5

2

Brevifolin carboxylic acid

8 DW content mg/g

DW content mg/g

14 DW content mg/g

DW content mg/g

2.0 DW content mg/g

DW content mg/g

25

16

Chlorogenic acid

Kaempferol glycoside

Kaempferol galloylglycoside

1

0

0 Date

0

Date

Date

Figure 4. The seasonal variation of some flavonol glycosides and phenolic acids in G. sylvaticum leaves from four populations: Contents are averages of all sized leaves in all four populations with standard deviation.


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35

Figure 5. Variation in contents of A) geraniin, B) carpinusin, C) PA total and D) PC% between populations in roots of G. sylvaticum.



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36

Figure 6. The ontogenic phases of G. sylvaticum flowers and the simplified ontogenic trend of polyphenols.


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37 SCHEMES

Scheme 1. Example structures of phenolic acids, proanthocyanidins, flavonol glycosides, galloylglucoses and gallotannins, and the simplified biosynthetic route of galloylglucoses to gallotannins.



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38

Scheme 2. Structures of ellagitannins found in G. sylvaticum and their proposed structural relations.


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39

Scheme 3. Structures and the proposed biosynthetic route of sylvatiins, and the main anthocyanin found in the petals of G. sylvaticum.



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40 TABLES Table 1. Quantified phenolic acids and flavonoids from G. sylvaticum, their molecular weight, MRM methods used, retention times and standard compounds used. compound MW transition CV /V CE /V RT/min standard used phenolic acids tryptophan 204.09 202.80 → 115.80 20 20 2.63 isolated from G. sylvaticum seeds9 brevifolin carboxylic acid 292.02 291.00 → 247.00 40 20 3.23 as ellagic acid equivalents monoG shikimic acid 326.06 325.00 → 169.00 20 20 2.19, 2.26 isolated from G. sylvaticum leaves9 monoG quinic acida 344.08 343.00 → 191.00 38 20 1.81, 1.92 isolated from G. sylvaticum roots9 chlorogenic acid 354.10 353.20 → 191.00 20 20 2.93 ExtraSynthese (Genay, France) diG quinic acid 496.09 495.00 → 343.00 40 20 2.69, 2.72, 2.80 isolated from G. sylvaticum roots27 flavonoids kaempferol 3-glucoside 448.10 447.18 → 285.00 40 25 4.29, 4.40 ExtraSynthese (Genay, France) quercetin 3-glycoside 464.10 463.16 → 300.00 35 30 4.05, 4.09 ExtraSynthese (Genay, France) kaempferol G-glycoside 600.11 599.00 → 285.00 50 30 4.42, 4.50 isolated from G. sylvaticum leaves9 as kaempferol G-glycoside kaempferol diglycoside 610.15 609.31 → 285.00 50 35 3.65, 3.86 equivalents quercetin G-glycoside 616.11 615.00 → 301.00 54 40 4.10, 4.15 isolated from G. sylvaticum leaves9 quercetin diglycoside 626.15 625.00 → 300.00 50 30 3.58 as quercetin glycoside equivalents malvin chloride 691.03 671.20 → 509.20 20 20 2.84 br, 3.03 Extrasynthese (Genay, France) malvin acetyglucoside 697.20 713.20 → 551.10 20 20 3.55 br, 3.68 as malvin equivalents PC dimer (total of isomers) 578.14 577.00 → 125.00 35 35 3.26, 2.96 std isolated from pine bark28 PD dimer (total of isomers) 610.13 609.00 → 125.00 25 30 2.13 isolated from G. sylvaticum roots a quantified using UV detection G, galloyl; MW, molecular weight; CV, cone voltage; CE, collision energy; RT, retention time; PC, procyanidin; PD, prodelphinidin


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41 Table 2. Quantified hydrolyzable tannins from G. sylvaticum, their molecular weight, MRM methods used, retention times and standard compounds used. compound MW transition CV /V CE /V RT/min standard used monoG glucose 332.08 331.00 → 169.0029 34 22 1.24, 1.47 isolated from G. sylvaticum roots27 methylated monoG glucose 346.08 345.00 → 183.00 20 20 2.64 as monoGG equivalents diG glucose 484.09 483.00 → 169.00 45 25 2.25, 2.66, 2.81 as diG quinic acid equivalents methylated diG glucose 498.09 497.00 → 345.00 20 20 3.38 as diG quinic acid equivalents triG glucose 636.10 635.00 → 465.0029 32 24 3.37 as diG quinic acid equivalents 788.10 787.20 → 169.00 60 35 3.70, 4.03, 4.10 as pentaG glucose equivalents tetraG glucose pentaG glucose 940.12 939.10 → 169.0030 60 32 4.37 synthetized from tannic acid pentaG glucose (metadepside) 940.12 939.10 → 787.00 40 25 4.42 br as pentaG glucose equivalents heksaG glucose 1092.13 1091.28 → 787.00 40 30 4.73 br as pentaG glucose equivalents heptaG glucose 1244.14 621.10 → 169.00 20 40 5.03 br as pentaG glucose equivalents G-acetyl-glucose 374.09 373.00 → 169.00 34 22 2.64 as sylvatiin A equivalents 29 G-HHDP-glucose A 634.08 633.00 → 301.00 46 32 3.11 as DiG-HHDP-glucose equivalents G-HHDP-glucose B 634.08 633.00 → 301.0029 46 32 3.26 as DiG-HHDP-glucose equivalents diG-HHDP-glucose 786.09 785.00 → 301.00 50 40 3.69 isolated from G. sylvaticum roots27 sylvatiin E 840.16 839.00 → 373.00 50 35 3.87 as sylvatiin A equivalents triG-HHDP-glucose 938.10 937.10 → 301.00 60 35 4.27 as carpinusin equivalents tellimagrandin II 938.10 937.20 → 301.00 60 35 3.82 as carpinusin equivalents geraniin 952.08 951.10 → 933.10 40 25 3.42 isolated from G. sylvaticum leaves9 carpinusin 952.08 951.10 → 933.10 40 25 4.16 isolated from G. sylvaticum leaves9 geraniinic acid 952.08 907.00 → 291.00 45 35 3.22 as carpinusin equivalents chebulagic acid 954.10 953.00 → 301.00 55 35 3.93 as carpinusin equivalents G-HHDPDHHDPmodified-glucose 970.09 969.10 → 925.10 55 25 3.37 as carpinusin equivalents 992.16 495.30 → 169.00 20 30 4.18, 4.25 as sylvatiin A equivalents sylvatiin B ascorgeraniin 1110.10 1109.20 → 933.00 60 35 3.54 as carpinusin equivalents sylvatiin A 1144.17 571.20 → 169.00 30 25 4.43, 4.46 isolated from G. sylvaticum petals11 sylvatiin D 1160.17 579.20 → 169.00 30 25 4.32 isolated from G. sylvaticum petals11 sylvatiin C 1348.24 673.30 → 169.00 30 35 4.49 isolated from G. sylvaticum petals11 MW, molecular weight; CV, cone voltage; CE, collision energy; RT, retention time G, galloyl; HHDP, hexahydroxydiphenoyl; DHHDP, dehydrohexahydroxydiphenoyl; MW, molecular weight; CV, cone voltage; CE, collision energy; RT, retention time ACS Paragon Plus Environment


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42 Table 3. The seasonal variation of polyphenols in the leaves of G. sylvaticum. month May June July n 60 60 36 compound content mg/g content mg/g content mg/g geraniin 142.6 ± 15.6 a 148.2 ± 20.1 a 159.5 ± 22.0 b carpinusin 33.9 ± 10.1 a 35.1 ± 14.5 a 41.5 ± 15.7 ab G-HHDP-glucose B 11.1 ± 4.1 a 16.5 ± 6.1 a 22.4 ± 8.0 b 1,2-diG-4,6-HHDP-glucose 1.5 ± 0.2 a 2.1 ± 0.3 b 2.4 ± 0.5 c triG-HHDP-glucose 2.9 ± 2.4 a 1.7 ± 1.3 b 1.9 ± 1.6 ab geraniinic acid 4.0 ± 2.1 a 4.0 ± 1.3 a 4.4 ± 1.8 a chebulagic acid 3.1 ± 1.1 a 7.7 ± 2.6 b 10.0 ± 3.7 c ascorgeraniin 2.0 ± 1.1 a 4.5 ± 1.5 b 4.5 ± 2.1 b monoG glucose 27.3 ± 23.5 a 6.9 ± 8.6 b 4.4 ± 3.3 b diG glucose 10.0 ± 5.8 a 5.1 ± 3.2 b 5.4 ± 3.2 b triG glucose 6.1 ± 3.9 a 2.4 ± 1.8 b 2.1 ± 1.3 b tetraG glucose 13.1 ± 8.4 a 6.1 ± 4.7 b 6.0 ± 4.2 b pentaG glucose 1.7 ± 1.9 a 0.2 ± 0.4 b 0.2 ± 0.3 b monoG quinic acid 10.5 ± 5.5 9.1 ± 4.0 8.9 ± 3.7 monoG shikimic acid 18.7 ± 7.0 a 13.2 ± 4.1 b 8.5 ± 2.8 b chlorogenic acid 14.3 ± 7.3 17.2 ± 6.6 15.4 ± 7.0 brevifolin carboxylic acid 6.3 ± 1.8 a 6.5 ± 1.1 a 7.0 ± 2.0 ab quercetin 3-glycoside 5.4 ±1.6 a 4.5 ± 1.6 b 3.5 ± 1.6 c quercetin G-glycoside 50.5 ± 9.0 a 45.3 ± 7.1 b 38.0 ± 7.2 c quercetin diglycoside 3.6 ± 2.0 a 3.0 ± 1.8 a 1.9 ± 1.2 b kaempferol 3-glycoside 1.2 ± 0.2 1.2 ± 0.2 1.3 ± 0.3 kaempferol G-glycoside 16.1 ± 7.1 15.9 ± 6.6 17.3 ± 5.9 a *0.01

Hydrolyzable Tannins, Flavonol Glycosides, and Phenolic Acids Show

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