Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show co-pigment effect

Phytochemistry xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show co-pigment effect Anu Tuominen ⇑, Jari Sinkkonen, Maarit Karonen, Juha-Pekka Salminen Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, FI-20014, University of Turku, Finland

a r t i c l e

i n f o

Article history: Received 6 August 2014 Received in revised form 10 January 2015 Available online xxxx Keywords: Geranium sylvaticum Geraniaceae Wood cranesbill ESI-MS/MS NMR Circular dichroism Protein precipitation capacity Co-pigmentation in the flower color manifestation Galloyl glucoses Ellagitannins

a b s t r a c t Four hydrolysable tannins, named as sylvatiins A (1), B (2), C (3) and D (4), were isolated from the petals of Geranium sylvaticum. On the basis of spectrometric evidence of NMR analysis (1H NMR, 13C NMR, DQF-COSY, TOCSY, NOESY, HSQC and HMBC), circular dichroism (CD) and ESI-MS/MS, sylvatiins A, B and C were characterized as galloyl glucoses containing one or two acetylglucoses attached to the 3-OH of the galloyl group, whereas sylvatiin D was found to have a chebulinic acid core containing acetylglucose attached in a similar way. The potential of these compounds to act as defensive compounds against herbivores was evaluated using the radial diffusion assay that measures the protein precipitation capacity. In addition, the capacity of sylvatiins to act as co-pigments with anthocyanins of G. sylvaticum petals was measured in vitro at different pH values. Sylvatiins A and D maintained efficiently the purple flower color near the natural pH of petal cells. The amount of sylvatiins was changed according to the flower color; deep purple petals with higher amount of anthocyanin contained more sylvatiins A and C than whiter petals. It was concluded that G. sylvaticum petal cells may accumulate sylvatiins for intermolecular co-pigmentation purposes. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Geranium sylvaticum (i.e. wood cranesbill) belongs to the family of Geraniaceae. It grows wild in most parts of Europe and is a common plant in Finland. It is a perennial herb that occurs in meadows, roadsides and herb-rich forests. Individual plant may have a few or even hundreds of flowers during flowering in June and each flower have five petals (Toivonen and Mutikainen, 2012). The color of petals can vary from white to deep purple; however white flowers are quite rare and more common to northern areas of Finland (Toivonen and Mutikainen, 2012; NatureGate, 2014). The main anthocyanin in G. sylvaticum that produces petal color is malvidin 3-(6-acetylglucoside)-5-glucoside (Andersen et al., 1995) (i.e. acetylmalvin). The phytochemistry of G. sylvaticum is interesting because it is rich in tannins and phenolic constituents which vary significantly in different organs (Tuominen, 2013; Tuominen et al., 2013). It contains both proanthocyanidins and three subgroups of hydrolysable tannins: galloyl glucoses, gallotannins and ellagitannins. The petals accumulated compounds that were not detected in the other ⇑ Corresponding author. Tel.: +358 2 3336828. E-mail address: ankatu@utu.fi (A. Tuominen).

organs. The UV spectra of these petal compounds resembled to those of galloyl glucoses, but molecular masses did not match any known hydrolysable tannin. In this work, four (1–4) of these novel galloyl derivatives were isolated, the exact structures and the location of additional acetylglucose (C8H14O7) moieties were confirmed and these compounds were named as sylvatiins A, B, C and D. Sylvatiins A, B and C (1, 2 and 3, respectively) were characterized as galloyl glucoses containing one or two acetyl glucose moieties attached to the phenolic hydroxyls of galloyl groups. Sylvatiin D (4) was observed to belong to the ellagitannins containing the chebuloyl group. It is not yet known, what is the ecological role of these compounds in the petals of G. sylvaticum. The petals of G. sylvaticum are heavily consumed by specialized insects (Asikainen and Mutikainen, 2005). Plants may accumulate secondary metabolites, such as astringent phenolic compounds as deterrence against insect herbivores. Here we studied the antiherbivore activity of sylvatiins in a relation to their protein precipitation capacity. Another role of sylvatiins may be related to the main function of petals, which is the attraction of pollinators with the bright purple color. Anthocyanins are usually colorless in plant cells’ vacuolar pH and therefore other phenolic compounds are needed for co-pigments to produce all the blue and purple colors of flowers (for example

http://dx.doi.org/10.1016/j.phytochem.2015.01.005 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

2

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

Asen et al., 1972; Yoshida et al., 2009). There is a previous study about the petals of some blue geraniums which found out that the pH value of vacuoles is near to 6 and flavonoids are used as co-pigments to maintain the blue color (Markham et al., 1997). The restricted appearance of sylvatiins only in the G. sylvaticum petals, and the fact that the main anthocyanin, acetylmalvin, contains the same acetylglucose moiety as these new hydrolysable tannins, support the close relation of these compounds. In this study, we measured the ability of sylvatiins to maintain the purple color of the anthocyanin fraction of G. sylvaticum petal extract at different pH values.

2. Results and discussion 2.1. Isolation and identification of new compounds 1–4 In the present study four new hydrolysable tannins (Fig. 1) were isolated from the G. sylvaticum petal extract with Sephadex LH-20 fractionation and semipreparative HPLC over C18. Sylvatiin A (1) (Fig. 1) was isolated first and obtained as white powder. The UV spectrum of 1 was identical to pentagalloylglucose (see Sections 4.3.1. and 4.4.1.). The ESI-MS in the negative ion mode exhibited a molecular ion at m/z 1143.1626 [MH] and a

doubly charged molecular ion at m/z 571.0823 [M2H]2 of which average value gave the accurate molecular mass of 1144.1745 Da, compatible with the molecular formula of C49H44O32. Singly charged fragment ions were observed in the ESI-MS/MS at m/z 939 and 769 after the cleavage of acetylglucose moiety (206 Da) and the cleavage of the acetylglucosylated gallic acid (374 Da) group, respectively (Table 1 and Figs. 1 and S1). The acetylglucosylated gallic acid residue was also observed as a fragment ion at m/z 373 (Table 1). The easy cleavage of the acetylglucose moiety indicated that it is attached to the galloyl group and not positioned between the galloyl group and center glucose. Other fragment ions with a high intensity were observed in ESI-MS/MS at m/z 973 and 617 after the cleavage of gallic acid (170 Da) and galloyl (152 Da) together with acetylglucosylated gallic acid, respectively (Table 1 and Figs. 1 and S1). These are typical fragmentation patterns of galloyl glucoses (Salminen et al., 1999; Tuominen et al., 2013). The same fragmentation patterns were observed for doubly charged ions; however, less collision energy was needed for break down the doubly charged ions (10 eV). The proton spectra showed the presence of signals from two sugars and galloyl groups. The proton and carbon resonances of the center glucose and the galloyl moieties of compound 1 corresponded well with the carbon and proton resonances and the coupling constants of pentagalloylglucose, which was used as

Fig. 1. The structures of new compounds from the petals of Geranium sylvaticum with the most typical fragmentation pathways of the compounds 1 and 4.

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

3

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

Table 1 Characteristic fragment ions determined by ESI-MS/MS analysis of new compounds 1–4. Peak intensities in parentheses are presented for singly charged ions obtained with the collision energy of 45 eV and for doubly charged ions with 15 eV. The bolded values are those fragments that occur already at lower collision energies. Compound

Accurate mass, observed

Exact mass, calculated

Error (ppm)

m/z

m/z

Sylvatiin A (1)

1144.1745

1144.1816

6.19

1143 [MH] (21.1)a 973 [MGAH] (59.1) 939 [MAGluH] (22.0) 821 [MGAGH] (21.0) 769 [MAGluGAH] (100.0) 617 [MAGluGAGH] (50.8) ? 465 [MAGluGA2GH] (8.3) 447 [MAGlu2GAGH] (25.9) 431 [MAGlu3GA] (30.5) 373 [AGlu+GAH] (25.3) ? 313 ? 295 ? 127, 169

571 495 469 393

[M2H]2 (100.0)a [MG2H]2 (13.5) [MAGlu2H]2 (43.0) [MAGluGH]2 (7.5)

Sylvatiin B (2)

992.1626

992.1706

8.04

991 787 635 617 465 447 373 295

495 419 393 317 241

[M2H]2 (49.5)a [MGH]2 (50.7) [MAGluH]2 (100.0) [MAGluGH]2 (94.2) [MAGlu2GH]2 (6.5)

Sylvatiin C (3)

1348.2446

1348.2450

0.30

1347 [MH] (67.2)a 1143 [MAGluH] (25.8) 973 [MAGluGAH] (100.0) 821 [MAGluGAGH] (16.7) 769 [M2AGluGAH] (48.5) 617 [M2AGluGAGH] (9.7) 447 [M2AGlu2GAGH] (0.0) ? 277, 295 431 [M2AGlu3GA] (0.0) ? 261 [M2AGlu4GA]– 373 [AGlu+GAH] (9.0)

673 597 571 495 469 373

[M2H]2 (100.0)a [MGH]2 (2.7) [MAGluH]2 (20.1) [MAGluG2H]2 (1.3) [M2AGlu2H]2 (1.5) [AGlu+GAH] (7.9)

Sylvatiin D (4)

1160.1699

1160.1765

5.67

1159 [MH] (50.0)a 1007 [MGH] (12.1) 989 [MGAH] (23.0) 955 [MAGluH] (11.4) 821 [MCheH] (26.1) 669 [MCheGH] (27.9) ? 465 [MCheGAGluH] (52.4) 651 [MCheGAH] (19.5) ? 499 [MCheGAGH] (24.3) 617 [MCheAGluH] (25.9) 373 [AGlu+GAH] (28.1) 337 [CheH] (77.9) ? 319 [CheH2OH] (70.0) ? 275 [CheH2OCOOH] (100.0) 293 [CheCOOH] (43.0) ? 247 [CheCOOCOOHH] (17.5)

579 557 477 455

[M2H]2 (100.0)a [MCOOHH]2 (17.4) [MAGluH]2 (32.0) [MAGluCOOHH]2 (10.5)

[MH] (9.0)a [MAGluH] (100.0) [MAGluGH] (42.3) [MAGluGAH] (64.8) [MAGluGAGH] (66.3) ? 313 (12.3) [MAGlu2GAH] (20.2) [AGlu+GAH] (25.7) (20.4) ? 169, 127

AGlu = acetylglucose, G = galloyl, GA = gallic acid, Che = chebuloyl. a ion subjected for fragmentation.

a reference compound in NMR, and its literature values (Tables 2 and 3, Nishizawa and Yamagishi, 1982). The use of 1D-TOCSY confirmed the presence of two sugars and allowed the assignment of overlapping signals. The other glucose showed dH signals that were all at the lower ppm values which indicated that it is mainly free from substituents. On the contrary, the dC signals of this glucose were at higher ppm values than those of center glucose; the dC C-10 even at 103.89 ppm. After the identification of center glucose signals with the use of COSY and HSQC spectra, the HMBC long range correlations between the sugar protons and the carbons of ester bonds were used to determine the positions of galloyl groups (Fig. 2). Using this information about the carbonyl group, the HMBC provided a link to aromatic protons and then the other aromatic signals of galloyl groups could be assigned using the HMBC and HSQC spectra (Fig. 2). These signals at the aromatic region were partly overlapping. The attachment of acetylglucose to the hydroxyl group of galloyl affected so that two aromatic proton signals of the 6-galloyl group were split to two doublets having a coupling constant of 2 Hz due to the asymmetry (Table 3). Also the carbon signals of C-2 and C-6 of the 6-galloyl group were at different values at dC 112.13 and dC 113.44 compared with the corresponding one two-fold signal of pentagalloylglucose at dC 110.10

(Table 2). Signals corresponding with the presence of the acetyl group were observed at dC 171.53 (carbonyl) and at dC 20.76 and dH 2.01 (methyl). The HMBC long range correlations confirmed that the acetyl group was attached to the C-60 of extra glucose and that the extra glucose was attached via C-10 to the hydroxyl of the galloyl group in C-3 (Fig. 2). The CD spectra of 1 and other galloyl glucose type sylvatiins were similar to that of pentagalloylglucose (Fig. 3A). Galloyl glucoses show split-type Cotton effects induced by the interaction between adjacent galloyl groups (Okuda et al., 1982a) as in pentagalloylglucose; positive Cotton effect at 272 and negative at 299 nm (Fig. 3A). Characteristic crossovers, where the CD signals were zero, were at 220 nm and 283 nm approximately at the same wavelengths as the UV maxima of these compounds exist (see 4.3.1). The dipole–dipole coupling of two chromophores spatially close to each other can yield this kind of an exciton splitting of absorption to two narrow bands with opposite signs centered on UV maxima (Lightner, 1994). The measured values for 1 [h]  104 in MeOH 1.5 (273 nm), 0.05 (243 nm), 1.4 (227 nm) were similar to the Cotton effect values measured for pentagalloylglucose (see 4.4.1) and earlier reported for pentagalloylglucose in the literature [h]  104 in MeOH: 1.5 (270 nm), 0.2 (240 nm) and 1.2 (225 nm)

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

4

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

Table 2 C NMR chemical shifts (ppm) for pentagalloylglucose (PGG) and new compounds 1–4 in acetone-d6 at 298 K. 13

Position

PGG

Center glucose 1 2 3 4 5 6a 6e

93.32 71.74 73.29 69.27 73.95 62.80 62.80

93.32 71.80 73.28 69.42 73.89 63.02 63.02

93.44 73.90 74.05 71.88 73.37 63.48 63.48

93.63 71.83 73.08 69.50 73.99 63.10 63.10

92.16 71.17 62.10 69.02 75.56 65.09 65.09

1-O-galloyl 1A 2 3, 5 4A 5A 6 7A

119.90 110.36 146.03 139.82 146.03 110.36 164.98

119.91 110.37 146.00 139.80 146.00 110.37 164.96

120.14 110.32 146.17 139.62 146.17 110.32 165.09

119.92 111.27 146.15 141.46 146.46 113.36 164.71

120.11 110.60 146.25 139.86 146.17 110.60 164.93

2-O-galloyl 1 2 3 4 5 6 7

120.52 110.16 145.95 139.33 145.95 110.16 165.71

120.53 110.16 146.15 139.31 146.15 110.16 165.71

121.30 110.32 146.02 139.02 146.02 110.32 165.98

120.41 110.23 145.98 139.40 145.98 110.23 165.81

Chebuloyl 119.26 117.24 146.56 139.52 141.42 116.23 165.20

3-O-galloyl 1 2, 6 3, 5 4 7

120.50 110.10 145.88 139.17 165.93

120.61 110.30 145.95 139.38 165.93

120.58 110.13 145.85 139.19 165.90

120.33 110.55 146.05 139.72 164.91

4-O-galloyl 1 2 3 4 5 6 7

120.61 110.26 146.00 139.38 146.00 110.26 165.64

120.53 110.14 146.19 139.18 146.19 110.14 165.71

121.28 110.18 145.95 139.11 145.95 110.18 165.92

120.52 110.32 145.96 139.37 145.96 110.32 165.74

Chebuloyl 169.64 66.76 41.32 39.55 30.00 172.74 173.78

6-O-galloyl 1 2 3 4 5 6 7

121.40 110.10 146.15 139.04 146.15 110.10 166.39

121.43 112.13 146.51 141.52 145.85 113.44 166.09

121.55 112.21 146.08 141.43 146.42 113.48 166.17

121.44 112.17 146.20 141.69 146.55 113.46 166.10

121.14 111.85 146.17 141.16 146.38 113.33 166.19

103.89 74.69 77.07 71.30 75.21 64.21 64.21 171.53 20.76

103.99 74.65 77.13 71.31 75.20 64.20 64.20 171.38 20.72

103.83 74.66 77.08 71.44 75.20 64.21 64.21 171.38 20.74

103.80 74.63 77.00 71.37 75.11 64.24 64.24 171.50 20.77

Acetylglucose in 6-O-galloyl 10 20 30 40 50 6a0 6e0 70 acetyl C@O 80 acetyl Me Acetylglucose in 1-O-galloyl 100 200 300 400 500 6a00 6e00 700 acetyl C@O 800 acetyl Me

1

2

3

4

102.99 74.51 77.11 71.31 75.04 64.32 64.32 171.45 20.95

(Okuda et al., 1982a). The similarity of Cotton effects of 1 and pentagalloylglucose indicated that the chromophores and their orientations were the same. Thus, sylvatiin A (1) was identified

as 6-O-(3-(6-acetyl)-glucosyl)-galloyl-1,2,3,4-tetra-O-galloyl-b-Dglucopyranoside. Sylvatiin B (2) (Fig. 1) was obtained as light yellow powder. It was assigned to have a molecular formula of C42H40O28 from the singly and doubly charged molecular ions observed in the ESI-MS spectrum at m/z 991.1558 [MH] and 495.0738 [M2H]2, which yielded the average accurate molecular mass of 992.1626 Da. Fragmentation patterns observed in ESI-MS/MS were similar to those of 1 and indicated that the difference between these compounds was that 2 had one galloyl group less. The position of the free hydroxyl group in the sugar ring was confirmed with NMR to be at C-3. The chemical shift of C-3 of center glucose, which had a hydroxyl group instead of a galloyl moved to lower frequency compared with 1 or 3 (Table 3). In contrast, the chemical shifts of corresponding carbon and neighboring carbons moved to higher frequency (Table 2). No correlation was observed in the HMBC spectra between the proton H-3 and the carbonyls of galloyl groups. The NOESY spectra showed that the protons H-2 and H-3 of glucose had a stronger correlation signal when there was no galloyl group attached at C-3. The structure and the position of acetylglucose moiety attached to the 6-galloyl group were confirmed in the same way as for the 1. Thus sylvatiin B (2) was identified as 6-O-(3-(6-acetyl)-glucosyl)-galloyl-1,2,4-triO-galloyl-b-D-glucopyranoside. Sylvatiin C (3) (Fig. 1) was obtained as light pink powder. The doubly charged molecular ion at m/z 673.1144 was compatible with the molecular formula of C57H56O38 and indicated the accurate mass of 1348.2446 Da. The molecular mass and fragmentation patterns indicated that another acetylglucose unit was attached to the compound when compared with 1 (Table 1). The use of 1DTOCSY was possible because the signals of H-50 at dH 3.77 and H500 at dH 3.96 differed enough and enabled the assignation of all three sugars although the signals were overlapping. The position of this extra glucose was confirmed with the HMBC measurements to be at the 1-galloyl group. The attachment of another acetylglucose to hydroxyl groups of the 1-galloyl split the signals of aromatic proton to two doublets having the coupling constant of 2 Hz (Table 3). Otherwise the structure and the position of second sugar were identical to the sugar of the 6-galloyl group (Fig. 1). Thus the structure of sylvatiin C (3) was elucidated to be 1,6-O-di-(3-(6-O-acetyl)-glucosyl)-galloyl-2,3,4-tri-O-galloyl-bD-glucopyranoside.

Sylvatiin D (4) (Fig. 1) was obtained as light yellow powder. The singly and doubly charged molecular ions at m/z 1159.1598 and 579.0791, respectively, were compatible with the molecular formula of C49H44O33 and indicated the average accurate molecular mass of 1160.1699 Da. The UV spectrum of 4 differed from the UV spectra of other sylvatiins so that the valley between two absorption maxima was slightly less deep and the first absorption maximum moved from 218 nm to 220 nm (Section 4.3). These features are more characteristic for ellagitannins. And further, the mass spectral fragmentation showed that the structure of 4 differed from other sylvatiins (Table 1 and Fig. 1). Fragment ions that are typical of oxidized ellagitannins were observed, such as the ions after the cleavage of the acid group (44 Da) and the chebuloyl group (338 Da). The mass spectral data suggested that 4 contained chebulinic acid as a base structure. This observation was confirmed with NMR data. NMR shifts corresponded well with those reported for chebulinic acid (Pfundstein et al., 2010). The small vicinal JHH couplings (<4 Hz) of center glucose showed that the conformation of sugar is 1C4 with all ring protons equatorial instead of 4C1 with all axial protons observed for 1, 2 and 3 (Table 3). Typical resonances of the chebuloyl group which are easily distinguished from other sylvatiins were two additional carbonyl dC resonances at 172.74 and 169.64 ppm. In addition, two

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

Position

Pentagalloylglucose

Sylvatiin A (1)

Sylvatiin B (2)

Sylvatiin C (3)

Sylvatiin D (4)

m

J/Hz

d/ ppm

m

J/Hz

d/ ppm

m

J/Hz

d/ ppm

m

J/Hz

d/ ppm

m

J/Hz

Center glucose 1 6.34 2 5.62 3 6.02 4 5.67 5 4.57

d dd t t ddd

6.33 5.62 6.02 5.70 4.58

d dd t t m

d t t t t

d dd t t m

d m m d m

2.3 (1,2) 2.75 (1,2); 1.9 (2,3) 1.9 (3,2) 3.4 (4,5) Not resolved

4.40 4.58

dd m

4.31 4.52

dd dd

4.41 4.63

dd dd

8.3 (1,2) 9.8 (2,3); 8.3 (2,1) 9.8 (3,2); 9.7 (3,4) 9.7 (4,5); 9.9 (4,3) 9.9 (5,4); 4.9 (5,6a); 2.0 (5,6e) 13.0 (6a,6e); 4.9 (6a,5) 13.0 (6e,6a); 2.0 (6e,5)

6.53 5.49 6.37 5.11 4.75

dd dd

8.5 (1,2) 9.3 (2,3); 8.5 (2,1) 9.3 (3,2); 9.5 (3,4) 9.5 (4,3); 9.0 (4,5) 9.0 (5,4); 4.5 (5,6a); 3.5 (5,6e) 9.0 (6a,6e); 4.5 (6a,5) 9.0 (6e,6a); 3.5 (6e,5)

6.30 5.67 6.05 5.71 4.60

4.41 4.55

8.5 (1,2) 10.0 (2,3); 8.5 (2,1) 10.0 (3,2); 9.5 (3,4) 9.5 (4,5); 9.5 (4,3) 9.5 (5,4); 4.6 (5,6a); 2.0 (5,6e) 13.0 (6a,6e); 4.6 (6a,5) 13.0 (6e,6a); 2.0 (6e,5)

6.10 5.41 4.33 5.41 4.38

6a 6e

8.5 (1,2) 10.0 (2,3); 8.5 (2,1) 10.0 (3,2); 9.5 (3,4) 10.0 (4,5); 9.5 (4,3) 10.0 (5,4); 4.5 (5,6a); 2.0 (5,6e) 12.5 (6a,6e); 4.5 (6a,5) 12.5 (6e,6a); 2.0 (6e,5)

4.73 4.91

dd m

Not resolved Not resolved

1-O-galloyl 2 6

6.85 6.85

s s

7.12 7.12

s s

7.10 7.10

s s

7.38 7.29

d d

2.0 (2,6) 2.0 (2,6)

7.30 7.30

s s

2-O-galloyl 2 6

7.47 7.47

s s

7.01 7.01

s s

7.18 7.18

s s

7.04 7.04

s s

3-O-galloyl 2 6

7.21 7.21

s s

7.06 7.06

s s

7.01 7.01

s s

7.44

s

7.00

s

7.08

s

d/ ppm

4-O-galloyl 2 3 4 5a 5e 6

7.44

s

7.00

s

6-O-galloyl 2 6

7.55 7.55

s s

7.45 7.37

d d

Acetylglucose in 6-O-galloyl 10 20 30 40 50

4.93 3.57 3.65 3.48 3.80

d t t t m

6a0 6e0 80 acetyl Me

4.30 4.44 2.01

dd dd s

7.11

s

7.11

s

2.0 (2,6) 2.0 (2,6)

7.41 7.37

d d

8.0 (10 ,20 ) 9.0 (20 ,10 ); 9.0 (20 ,30 ) 9.0 (30 ,20 ); 9.0 (30 ,40 ) 9.0 (40 ,30 ); 9.0 (40 ,50 ) 9.0 (50 ,40 ); 7.0 (50 ,6a0 ); 2.0 (50 ,6e0 ) 12.0 (6a0 ,6e0 ); 7.0 (6a0 ,50 ) 12.0 (6e0 ,6a0 ); 2.0 (6e0 ,50 )

4.88 3.52 3.60 3.42 3.76

d t t t m

4.25 4.39 2.00

dd dd s

Chebuloyl 7.56 s

7.21 7.21

s s

Chebuloyl 4.97 d 5.19 dd 3.97 ddd 2.26 2.27

d d

7.2 (2,3) 7.2 (3,2); 1.5 (3,4) 1.5 (4,3); 3.5 (4,5a); 10.0 (4,5b) 3.5 (5a,4) 10.0 (5b,4)

7.08

s

2.0 (2,6) 2.0 (2,6)

7.44 7.38

d d

2.0 (2,6) 2.0 (2,6)

7.32 7.24

d d

2.0 (2,6) 2.0 (2,6)

8.0 (10 ,20 ) 8.5 (20 ,10 ); 8.5 (20 ,30 ) 9.5 (30 ,20 ); 9.5 (30 ,40 ) 9.5 (40 ,30 ); 9.5 (40 ,50 ) 9.5 (50 ,40 ); 7.0 (50 ,6a0 ); 2.1 (50 ,6e0 ) 12.0 (6a0 ,6e0 ); 7.0 (6a0 ,50 ) 12.0 (6e0 ,6a0 ); 2.1 (6e0 ,50 )

4.93 3.56 3.63 3.45 3.77

d dd t t ddd

4.89 3.53 3.63 3.44 3.80

d t t t m

4.23 4.39 2.01

dd dd s

8.0 (10 ,20 ) 9.5 (20 ,30 ); 8.0 (20 ,10 ) 9.0 (30 ,20 ); 9.0 (30 ,40 ) 8.5 (40 ,30 ); 8.5 (40 ,50 ) 9.5 (50 ,40 ); 6.5 (50 ,6a0 ); 2.0 (50 ,6e0 ) 12.0 (6a0 ,6e0 ); 7.5 (6a0 ,50 ) 12.0 (6e0 ,6a0 ); 2.0 (6e0 ,50 )

4.26 4.40 2.02

dd dd s

8.0 (10 ,20 ) 9.0 (20 ,10 ); 9.0 (20 ,30 ) 9.0 (30 ,20 ); 9.0 (30 ,40 ) 9.0 (40 ,30 ); 9.0 (40 ,50 ) 9.0 (50 ,40 ); 7.0 (50 ,6a0 ); 2.0 (50 ,6e0 ) 12.0 (6a0 ,6e0 ); 7.0 (6a0 ,50 ) 12.0 (6e0 ,6a0 ); 2.0 (6e0 ,50 )

Acetylglucose in 1-O-galloyl 100 200 300 400 500

4.94 3.54 3.66 3.44 3.96

d dd t t ddd

6a00 6e00 800 acetyl Me

4.27 4.46 2.03

dd dd s

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

7.9 (100 ,200 ) 9.2 (200 ,300 ); 7.9 (200 ,100 ) 9.2 (300 ,200 ); 9.2 (300 ,400 ) 9.3 (400 ,300 ); 9.3 (400 ,500 ) 9.85 (500 ,400 ); 6.9 (500 ,6a00 ); 2.0 (500 ,6e00 ) 11.8 (6a00 ,6e00 ); 6.9 (6a00 ,500 ) 11.8 (6e00 ,6a00 ); 2.0 (6e00 ,500 )

5

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

Table 3 H NMR data for pentagalloylglucose and new compounds 1–4 in acetone-d6 at 298 K.

1

6

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

Fig. 2. Important HMBC correlations for sylvatiin A (1) and sylvatiin D (4).

A

4 3

[ ] x 10-4M

2

Sylvatiin A

1 0

Sylvatiin B

-1

Sylvatiin C

-2 Pentagalloyl glucose

-3 -4 200

250

300 350 Wavelength (nm)

400

[ ] x 10-4M

BB 10 8 6 4 2 0 -2 -4 -6 -8

Sylvatiin D Chebulinic acid Chebulanin

200

250

300 350 Wavelength (nm)

400

Fig. 3. The CD spectra of (A) galloyl glucoses and (B) ellagitannins in MeOH.

aliphatic C-5 protons at 2.26 ppm were easily observed (Table 3). Long-range couplings in the HMBC spectra were observed throughout the chebuloyl group as presented in Fig. 2. The strong NOE confirmed close distance between H-20 and H-30 of the chebuloyl

group. The arrangement of H-20 and H-30 , and H-30 and H-40 in the chebuloyl group were consistent with the observed vicinal coupling constants J20 ,30 of 7.3 Hz and J30 ,40 of 1.5 Hz and literature values (Pfundstein et al., 2010). The configuration of the chebuloyl group was confirmed with CD spectra by comparing with the spectra of purified reference compounds that contain the same group (Fig. 3B). The compound 4 and chebulinic acid showed a small peak at 300 nm, opposite to the Cotton effect of chebulanin, pentagalloylglucose and other sylvatiins, presumably as a result of the different orientation of galloyl chromophores due to the different glucopyranose conformation (Fig. 3). The Cotton effects of 4 [h]  104 in MeOH (nm) +5.6 (218), 1.3 (242), 0.6 (256), 0.8 (269), +0.7 (298) were similar to corresponding values of chebulinic acid [h]  104 in MeOH (nm) +6.6 (218), 2.0 (240), 0.1 (260), 0.2 (273), +1.3 (297). Only the intensities were lower for compound 4 maybe as a result of to the acetylglucose group attached in the 6-galloyl. However, the Cotton effects of 4 indicated the same configuration (2S, 3S, 4S) for the chebuloyl group as observed and reported for chebulanin and chebulinic acid (Pfundstein et al., 2010; Yoshida et al., 1982). Thus, sylvatiin D (4) was identified as 6-O-(3-(6-acetyl)glucosyl)-galloyl-1,3-di-O-galloyl-2,4-(20 S,30 S,40 S)-chebuloyl-b-Dglucopyranoside. We have not found previous studies reporting that galloyl groups of hydrolysable tannins could exist as glycosylated derivatives in a similar manner as flavonoids and other polyphenols. However, Tanaka et al. (1992) have reported dehydroellagitannin-acetone condensates with similar masses 840 Da, 992 Da and 1160 Da, which were observed for sylvatiins in the present study. Those compounds were artifacts from the isolation with hot acetone. We confirmed by the extraction with pure water and methanol that the acetyl moiety of sylvatiins is not an artifact of acetone used as an extraction solvent. Moreover, the NMR results unequivocally confirmed that structures are as presented, that the acetyl group is attached to additional glucose and compounds do not contain dehydrohexahydroxydiphenyl (DHHDP) groups where acetyl could be attached. This was also consistent with the characteristic fragment ions for acetylglucosylated hydrolysable tannins; the cleavage of acetylglucosylated galloyl group or acetylglucose moiety alone. Interestingly this acetylglucose part was found in both the 1C4 and 4C1 conformation type of hydrolysable tannins. Petals contained also smaller amounts of isomers of the isolated compounds. In addition, the petals of G. sylvaticum contained more minor compounds belonging to sylvatiin family with molecular masses of 374 Da, 840 Da (sylvatiin E), 1178 Da (Tuominen et al., 2013) and 1364 Da. Based on the HPLC-ESI-MS analysis and the data of isolated sylvatiins, the structures of these compounds can be concluded to have an acetylglucose attached to gallic acid, to trigalloyl glucose, to ellagitannin where one water molecule is added to the sylvatiin D structure (such as the dehydrochebuloyl group) and to sylvatiin D with the additional acetylglucose group, respectively. It is likely that the main isomers of these compounds follow the same substitution pattern as the isolated sylvatiins: the first acetylglucose is attached to the 6-galloyl group and the second one to the 1-galloyl group. 2.2. Biological role 2.2.1. Antiherbivore activity The ecological role of these compounds in the petals of G. sylvaticum is so far unknown. The petals of G. sylvaticum are often heavily consumed by herbivores (Asikainen and Mutikainen, 2005). The relatively high sugar content and the softness of petals compared with other G. sylvaticum flower parts might make them a preferred food source for herbivores (Tuominen, 2013). Traditionally, tannins are considered as the defensive compounds of plants

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

7

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

against herbivores and pathogens. There are two different hypotheses about the mechanism of this antiherbivore action. Defensive action can be due to the protein precipitation capacity that reduces the palatability of plant to herbivores as a food source, or a newer hypothesis assumes that the auto-oxidative conditions in the herbivores’ gut enable harmful pro-oxidant action (Appel, 1993; Salminen and Karonen, 2011). Galloyl glucoses have low or intermediate pro-oxidant activity and are less effective pro-oxidants than for example ellagitannins (Barbehenn et al., 2006a,b; Tuominen, 2013). It has been previously measured that the prooxidant activity of petal fractions containing sylvatiins is similar as for the pistil fractions containing galloyl glucoses (Tuominen, 2013). The addition of acetylglucose moiety to the galloyl glucose type structure did not seem to increase the pro-oxidant activity of sylvatiins significantly (Tuominen, 2013). The protein precipitation capacity is a typical property of tannins and is considered to be the underlying mechanism for example in astringency, fungitoxicity and enzyme inhibition (Kawamoto et al., 1996). Tannins have moderate affinities for BSA (Hagerman, 1987) however; the BSA protein precipitation capacity of pentagalloylglucose is the highest in a galloyl glucose series (Kawamoto et al., 1996). In this study, we measured the protein precipitation capacity of sylvatiins compared with the capacity of pentagalloylglucose by using BSA as a model protein. The radial diffusion assay (RDA) test showed that the addition of one acetylglucose to a galloyl group slightly decreased the protein precipitation capacity of sylvatiin A (1) compared with pentagalloylglucose, which has the same core structure (Table 4). Moreover, the addition of another acetylglucose to the 1-galloyl group (3) diminishes the protein precipitation capacity over 30%. This capacity loss was equal to the effect of the loss of one galloyl group (2) or the transformation of two galloyl groups to ellagitannin group such as the chebuloyl group of the compound 4 (Table 4). It is known that the steric hindering of galloyl groups to each other affects their protein precipitation capacity and at least three synergistically acting galloyl groups are required for effective complexation (Kawamoto et al., 1995, 1996). Tanaka et al. (1997) have observed that a galloyl group at the anomeric C-1 position makes the largest contribution to the hydrophobic interaction between pentagalloylglucose and other compounds. It seems that acetylglucose shields the galloyl group binding site from the interaction with the protein and especially the acetylglucose attached to the 1-galloyl group as in 3 seems to have stronger effect to the protein precipitation capacity of sylvatiins than the acetylglucose in the 6-galloyl group (Table 4). Pentagalloylglucose is the most hydrophobic of galloyl glucoses and forms a gel with water in high concentrations (Tanaka et al., 1997). Chebulinic acid, the other core compound of sylvatiins, is

also poorly water-soluble. Additional acetylglucose group may enhance the water-solubility of these compounds as sugars typically do, so that higher concentrations of sylvatiins can be accumulated to the vacuole cell sap. At the end, sylvatiins were quite effective in protein precipitation assay and might be accumulated into petal cells for those purposes. The live-span of G. sylvaticum petals is short, the flowers remain open two to three days (Varga et al., 2013), and thus the deterrence of petals against herbivores may not be a very important feature for plant fitness. 2.2.2. Co-pigmentation The purple and blue colors of petals which attract pollinators are produced by anthocyanin pigments. However, many petal cells have pH where anthocyanins are practically colorless; the vacuolar pH of plants is typically 5.0–5.5, but some purple flowers can have pH over 6 (Taiz, 1992). The vacuolar pH of the petals of G. sylvaticum was estimated from pressed juice to be 4.8 (Table 5). In previous study with some other geraniums with the blue petal color, pH above 6 was needed in epidermal cells to maintain the special color characteristics; this pH was higher than measured from the pressed juice (Markham et al., 1997). The stabilization of the colors of anthocyanins at these pH values can be achieved with a complexation of the flavylium cation and quinonoid bases by other polyphenols and/or metals. Co-pigmentation enhances the absorption intensity of pigment (hyperchromism) and shifts the absorption maximum to higher wavelengths (bathochromism) (Haslam, 1998). Kaempferol, myricetin and quercetin glycosides are known flavonoid co-pigments of anthocyanins in some blue geraniums (Markham et al., 1997). In addition, many colorless natural compounds such as hydroxylated benzoic and cinnamic acids, hydroxyflavones and tannins are able to act as co-pigments (Asen et al., 1972; Brouillard et al., 1989). Flavonol glycosides are usually the most effective co-pigments, for example quercetin-3galactoside increased the absorbance of malvin chloride at pH 3.65 by 173% (Mistry et al., 1991). Only a few studies have measured the co-pigmentation effect of hydrolysable tannins (Cai et al., 1990; Mistry et al., 1991). Ellagitannins and galloyl glucoses can easily be left unnoticed in earlier studies if for example only detection wavelengths between 300 and 600 nm, which are typical for flavonoids and anthocyanins, are used in the HPLC-DAD. This can be the reason for their neglecting in the petal color and co-pigmentation studies. The co-pigmentation effect was determined by a method of Brouillard et al. (1989) by calculating the relative absorbance change of the sample with co-pigment to the absorbance of the sample without co-pigment. Results in the Table 4 are presented at pH 5 for anthocyanin fraction because this pH is the nearest to

Table 4 Biological activity results of new compounds and the reference compounds used from the protein precipitation capacity and the co-pigmentation assays. Compound Sylvatiin A (1) Sylvatiin B (2) Sylvatiin C (3) Sylvatiin D (4) Sylvatiin E Geraniin Pentagalloylglucose Chebulinic acid Chebulagic acid Chebulanin

Molecular formula C49H44O32 C42H40O28 C57H56O38 C49H44O33 C35H36O24 C41H28O27 C41H32O26 C41H32O27 C41H30O27 C27H24O19

Molecular mass 1144 992 1348 1160 840 952 940 956 954 652

d2 (cm2) 1.28 ± 0.19 0.78 ± 0.06 0.97 ± 0.09 0.82 ± 0.04 0 1.05 ± 0.08 1.11 ± 0.07 1.15 ± 0.06 0

PPC % 12 43 32 34 100 29 0 18 20 100

(A  A0)/A0  100% at 550 nm at pH 5

(A  A0)/A0  100% at 520 nm at pH 4

Anthocyanin fraction 144.4 ± 0.7

Malvin chloride 197.6 ± 0.7

49.7 ± 0.3 169.9 ± 0.1 11.5 ± 0.3

62.9 ± 0.1 283.2 ± 0.1 19.9 ± 0.1

55.1 ± 0.7 55.4 ± 0.7

38.1 ± 0.1 69.9 ± 0.8

d2 = the square of the diameter of circle in the RDA assay, values shown are the average of three replicates ± s.d. PPC % = the protein precipitation capacity of compound compared to the capacity of pentagalloylglucose. (A  A0)/A0 = the co-pigmentation effect of anthocyanin and compound expressed as the relative absorbance of sample compared to blank, values shown are the average of three replicates ± s.d.

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

8

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

Table 5 Characteristics of different colored G. sylvaticum petals. Petal color

Deep purple

Medium purple

White

Deep purple

Sample type n pH

Dried and ground 10 5.0

Dried and ground 8 5.0

Dried and ground 13 nd

Pressed juice 1 4.8









Concentrations a

103 mol/g

103 mol/g

103 mol/g

103 mol/l

Acetylmalvin (5) Sylvatiin A (1)a Sylvatiin B (2)a Sylvatiin C (3)a Sylvatiin D (4)a Quercetin diglycoside (6)b Kaempferol diglycoside (7)b Kaempferol 3-glycoside (8)b Kaempferol galloylglycoside (9)b

41.8 26.1 9.0 36.7 25.6 16.2 37.3 15.3 23.5

14.7 18.8 7.6 25.9 27.1 16.7 81.3 28.5 50.3

1.8 14.5 8.5 14.4 25.0 23.3 92.8 30.6 46.3

4.6 0.5 0.2 1.6 1.7 0.8 2.9 0.8 1.0

Total sylvatiins Total flavonoids

97.4 92.4

79.4 176.8

62.4 193.0

4.0 5.5

Ratio acetylmalvin/sylvatiins Ratio acetylmalvin/flavonoids

1:2 1:2

1:5 1:12

1:35 1:107

1:1 1:1

nd = not determined. a Quantified using optimized SRM method. b Quantified using UV detection at wavelength 349 nm.

Fig. 4. The color manifestation of (A) malvin chloride and (B) the anthocyanin fraction from G. sylvaticum petals at different pH values with different co-pigments and an example of natural color of deep purple and medium purple flower. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

the measured vacuolar pH of G. sylvaticum and at pH 4 for malvin to ease the comparison to literature values. In previous studies, the core compound of sylvatiin B and C, pentagalloylglucose, was effective co-pigment in in vitro tests and increased the absorbance of malvin (malvidin-3,5-diglycoside) chloride by 121% at pH 3.65 (Cai et al., 1990) and by 69% at pH 6 (Brouillard et al., 1991). In this study, pentagalloylglucose increased the absorbance of malvin by 38% at pH 4 but sylvatiins were more effective (Table 4). Sylvatiin A and sylvatiin D showed the strongest co-pigmentation effect, 198% and 283%, respectively (Fig. 4 and Table 4). These two compounds were the most effective with both pigments tested and throughout the used pH scale (Figs. 4, S2 and S3). The color stability of malvin was measured separately after 1 h incubation and the absorbance of samples that contained added co-pigments was slightly increased in water and at pH 4 and 5 (Table S1). On the contrary, at higher pH values the absorbance of samples was decreased over 10% in all other samples except those that contained sylvatiin A or D (Table S1). It was unexpected that sylvatiin D was the most active co-pigment because it has been stated that ellagitannins are typically less efficient co-pigments than galloyl glucoses because the molecule structure is less flexible (Cai et al., 1990). Therefore, it should be further studied how the chebuloyl group interacts with anthocyanin. Interestingly, the addition of second acetylglucose to the 1galloyl did not enhance the co-pigmentation effect of 3 in a similar way that the protein precipitation activity was affected. The observed color of anthocyanin solution is dependent on the intensity of different absorption maxima that are caused by different forms of anthocyanin that coexist at various pHs as shown for malvin in Fig. 5A. When only water was added, the

Fig. 5. The UV spectra of malvin at different pH without co-pigment (A) and with sylvatiin D (B). The concentration of both compounds in the mixture was ca. 1 mM and the spectra were measured before 1 min after mixing.

9

pH of solution was 3.3 due to the acidity of malvin and the relatively sharp maximum (k1) of sole flavylium (reddish AH) at 520 nm was observed (Fig. 5A). After the buffer addition, an equilibrium mixture of free flavylium and neutral quinonoid forms (A, two possible tautomers) was formed and observed as a broad band around 530 nm. The absorption maxima of AH and A species are so close to each other that they cannot be distinguished (Nave et al., 2010). At pH 7 and 8, the absorption maximum k3 of the anionic quinonoid base (A) occurs as a broad band around 620 nm in the blue color region (Figs. 4 and 5A). However, the absorbance rise below 450 nm indicates that yellow colored chalcones are also formed (Fig. 5A) and then the mixture of these species are seen as a green color to the human eye (Fig. 4). The UV spectra in Fig. 5B show that sylvatiin D stabilizes both the flavylium cation (k1) and the neutral quinonoid (k2) species of malvin, which is observed as a broad band around 570 nm. The binding with co-pigment yields a bathochromic shift of ca. 40 nm due to the co-pigment-to-pigment charge transfer. In addition, the intensity of these maxima stayed at twice higher level when compared with intensities without co-pigment (Fig. 5). The measured spectra for the G. sylvaticum petal fraction with co-pigment and spectra with other co-pigments, such as sylvatiin A, were very similar to the presented spectra of malvin in Fig. 5B. Also previous studies have concluded that a similar spectrum with three absorption maxima at ca. 540 nm, 575 nm and 620 nm is typical for purple–blue flowers (Markham et al., 1997). Sylvatiins may have advances in their structure for co-pigmentation compared with regular galloyl glucoses. The complexation between anthocyanin and galloyl groups has been observed to take place mainly at galloyl ester groups at C-1 and C-6 which are maintained in the optimal position and separation for hydrophobic p–p stacking of aromatic nuclei (Cai et al., 1990). The same galloyl groups of sylvatiins contain additional acetylglucoses (Fig. 1). These acetylglucose groups may further stabilize the complex by providing more hydrogen bonding sites between hydroxy and carbonyl groups. The hydrophilic layer of sugar moieties can assist and stabilize the stacking by hydrogen bonding (Baranac et al., 1997; Goto and Kondo, 1991). Deep purple and white G. sylvaticum petal extracts had different phenolic profiles (Fig. 6). The amount of sylvatiins was dependent on the flower color; deep purple petals with high amount of anthocyanin contained one third more total sylvatiins than totally white petals (Table 5). Especially the content of sylvatiin A and C was

Fig. 6. The difference between phenolic profiles of extracts of deep purple and white petals of G. sylvaticum shown as TIC chromatograms. Other numbered compounds than sylvatiins are main anthocyanin acetylmalvin (5), four main flavonoids; quercetin diglycoside (6, 626 Da), kaempferol diglycoside (7, 610 Da), kaempferol 3-glycoside (8, 448 Da), kaempferol galloylglucoside (9, 600 Da).

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

10

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

correlated to the content of acetylmalvin. The content of sylvatiin B and D was more consistent. These compounds may be degradation and oxidation products; the content of sylvatiin D was significantly higher in samples that were air-dried compared with the freezedried samples. Instead of anthocyanin, white petals synthesized two times more flavonoids than purple petals (Fig. 6 and Table 5). The petal extracts of G. sylvaticum contained anthocyanins and sylvatiins in the molar ratio of 1:2, whereas the molar ratio in the pressed juice was 1:1 (Table 5). This can be due that part of the anthocyanins degraded in the extraction because conditions were not optimal for anthocyanins. However, the clear difference in the observed ratio between anthocyanin pigment and sylvatiins in the deep purple and whiter petals confirms the hypothesis of copigmentation (Table 5). Sylvatiins A and D have a strong bluing effect on the color manifestation of anthocyanin fraction from G. sylvaticum petals and maintain the purple color similar to the natural petal color at higher pH near to natural pH of petal cells (Fig. 4). Interestingly, the main anthocyanins in G. sylvaticum petals (Andersen et al., 1995) contain the same acetylglucose group as sylvatiins. In addition, sylvatiins did not significantly accumulate to other plant parts of G. sylvaticum than petals (Tuominen et al., 2013) and their content was following on the anthocyanin content. These results together indicate that G. sylvaticum petal cells may accumulate sylvatiins for intermolecular co-pigmentation purposes. 3. Conclusions Four novel hydrolysable tannins, sylvatiins A–D, were isolated from the petals of G. sylvaticum and their structures were characterized using mass spectrometry, NMR and CD spectroscopic methods. These sylvatiins contained an acetylglucose moiety attached to the hydroxyl groups of galloyl glucoses or chebulinic acid. The addition of acetylglucose group decreased the protein precipitation capacity of sylvatiins compared with galloyl glucoses. However, this modification may be useful in the enhancement of water-solubility so that these compounds can be accumulated to cells in effective amounts. Sylvatiins also showed the ability to maintain the purple color of petal anthocyanins in more neutral and mildly basic pH values. This and their occurrence only in petals strongly suggest that these compounds act as co-pigments. 4. Experimental 4.1. General experimental procedures The analyses of semipreparative fractions were performed with an ultra-high performance liquid chromatographic system (UHPLC, Acquity UPLCÒ, Waters Corporation, Milford, MA, USA) combined with a triple quadrupole mass spectrometer (XevoÒ TQ, Waters Corporation, Milford, MA, USA) and a diode array detector. An Acquity UPLCÒ BEH Phenyl (2.1  100 mm i.d., 1.7 lm, Waters Corporation, Wexford, Ireland) column was used. The ESI conditions and UPHLC methods were the same as described previously in Vihakas et al. (2014). The purity of compounds was measured with the HPLC-ESI-MS analysis using an Agilent 1200 Series HPLC system (Agilent Technologies, Waldbronn, Germany), which consisted of a binary pump, a degasser, a Hip-ALS SL autosampler, a DAD SL detector and a control module. Chromatographic separations were performed using a method previously described in Tuominen et al., 2013. Chromatograms were recorded at 280 nm. The HPLC system was controlled by Hystar software version 3.2. (Bruker BioSpin, Rheinstetten, Germany). HPLC was connected to a Bruker micrOTOF-Q ESI hybrid quadrupole with a time-of-flight mass spectrometer (Bruker Daltonics,

Bremen, Germany). The mass spectrometer was controlled by Compass micrOTOF control software (Bruker Daltonics) and operated in a negative ion mode. The capillary voltage was maintained at +4000 V with the end plate offset at 500 V. The pressure for the nebulizer gas (N2) was set at 1.6 bar and the drying gas (N2) temp. at 200 °C. The data were handled by Compass DataAnalysis software (version 4.0; Bruker Daltonics). The samples in MS/MS analysis were introduced by the direct infusion in a solution of MS grade MeOH at the flow rate of 240 ll/min. The mass scan ranged from m/z 40 up to 3000. The collision energy was changed from 0 to 100 eV with steps of 5 eV. NMR spectra were measured with a Bruker Avance 500 spectrometer (Fällanden, Switzerland). Purified compounds were dissolved to acetone-d6. The techniques used in structure elucidation were 1H NMR, 13C NMR, DQF-COSY, CH2-edited HSQC, HMBC, NOESY and 1D-TOCSY. The CD spectra were recorded with a Chirascan™ circular dichroism spectrometer (Applied Photophysics, Leatherhead, UK) and controlled by Chirascan Pro-Data software (Applied Photophysics, Leatherhead, UK). The samples were dissolved in MeOH and placed in an 1 cm cuvette. The spectra were scanned over the range of 200–400 nm at 22 °C. The data were handled by Applied Photophysics Pro-data Viewer (version 4.2.0, Applied Photophysics, Leatherhead, UK). The spectra were background subtracted and smoothed. The UV spectra of isolated compounds were recorded with a Lambda 25 UV/VIS spectrometer (PerkinElmer, Norwalk, USA). The data were handled by UV WinLab 6.0 software. The samples were dissolved in HPLC grade MeOH. 4.2. Plant material The G. sylvaticum petal material for compound isolation was collected from seven different populations around Finland during the summer of 2007. Voucher specimens (TUR 597241–597244) are deposited in the herbarium of the University of Turku. Petals were collected at the prime time of blooming in June and July and separated from other flower parts by hand. Material for petal color comparison was collected from Turku (eastern Finland), Orivesi (middle), and Savukoski (northern) during summers 2007 and 2014. Before freezing and drying, petal samples were visually divided into three color groups: deep purple (the most common), totally white and medium purple contained all other samples between first two groups. Fresh plant material for pH measurement was collected in September 2014 from Turku. The petals of ca. six flowers were crushed in Spin-X Centrifuge tube and filtered with the centrifuge through 0.22 lm Nylon. pH was measured from petal juice using Phenomenal pH 1000 L pH meter (VWR) with Biotrode (Hamilton, USA). 4.3. Extraction and isolation of new compounds 1–4 Collected petals were air- and freeze-dried and homogenized into fine powder. Several 200 mg samples of fine plant powder were extracted 4 times with 8 ml of acetone:water (7:3, v/v) with a planary shaker. Extracts from different populations were found to be quite similar by the HPLC analysis and therefore they were pooled together and concentrated into the aqueous phase by rotary evaporator at <40 °C. The aqueous phase was filtered to remove water insoluble compounds and thereafter the filtrate was freeze-dried. The total amount of 6.1 g of freeze-dried petal powder yielded a 3.6 g of freeze-dried petal extract. An aliquot of 3.1 g of freeze-dried extract was weighed and dissolved in the small amount of water for a column chromatography over Sephadex LH-20. After centrifuging, the clear sample soln. was applied to a Sephadex LH-20 column and 14 fractions of 500 ml were collected by elution with water (2) and 10%, 20%, 30%, 40% and 50% aq. MeOH, then 50% aq. MeOH and 10% aq. acetone

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

(2), 55% MeOH and 15% aq. acetone, and after that with 30%, 50%, 70% and 80% aq. acetone. The obtained fractions were concentrated into the aqueous phase by the rotary evaporator and freeze-dried. The purification of individual compounds was performed by Waters (USA) preparative HPLC consisting of a 2998 photodiode array detector, 600 controller and Delta 600 pump using a semipreparative Phenomenex Gemini RP-18 (150  21.20 mm i.d., 10 lm) column. The elution was conducted with varying gradients with acetonitrile/0.1% formic acid (0:100 ? 80:20, v/v) with the flow rate of 8 ml/min and 2 ml fractions were collected with a Waters fraction collector. The obtained fractions were analyzed with the UHPLC-DAD-MS and similar fractions were combined after the concentration into the aqueous phase by Eppendorf concentrator and freeze-dried. The Sephadex LH-20 fractionation yielded 199.7 mg of fraction eluted with 50% aq. MeOH and 10% aq. acetone. From this fraction, 23.0 mg (light pink powder, purity 93.7%) of compound 3 was achieved with the semipreparative column chromatography. From the next Sephadex LH-20 fraction (198.6 mg), eluted with 55% MeOH and 15% aq. acetone, 3.2 mg (light yellow powder, purity 80.6%) of compound 2 and 20.6 mg (light yellow powder, purity 85.7%) of compound 4 were achieved. The following Sephadex LH-20 fraction (55 mg) eluted with 30% aq. acetone was used for the purification of compound 1 (11.3 mg of white powder, purity of 73.4%). Fig. 1 shows the structures of isolated compounds. 4.3.1. 6-O-(3-(6-acetyl)-glucosyl)-galloyl-1,2,3,4-tetra-O-galloyl-b-Dglucopyranoside (1) Sylvatiin A, white powder; CD (MeOH) [h]215 22,734, [h]227 +13,964, [h]243 488, [h]273 +15,490, [h]299 11,485; UV (MeOH) kmax (log e) 218 (4.9), 280 (4.5) nm; 1H NMR see Table 3; 13C NMR see Table 2; HR-ESI-MS (negative ion mode): m/z 1143.1626 [MH] (calcd for C49H43O32, 1143.1738); ESI-MS/MS (negative ion mode) see Table 1. 4.3.2. 6-O-(3-(6-acetyl)-glucosyl)-galloyl-1,2,4-tri-O-galloyl-b-Dglucopyranoside (2) Sylvatiin B; light yellow powder; CD (MeOH) [h]216 14,320, [h]229 +11,000, [h]253 +4429, [h]268 +8633, [h]293 16,146; UV (MeOH) kmax (log e) 218 (5.0), 279 (4.6) nm; 1H NMR see Table 3; 13 C NMR see Table 2; HR-ESI-MS (negative ion mode): m/z 991.1558 [MH] (calcd for C42H39O28, 991.1628); ESI-MS/MS (negative ion mode) see Table 1. 4.3.3. 1,6-O-di-(3-(6-O-acetyl)-glucosyl)-galloyl-2,3,4-tri-O-galloyl-b(3) Sylvatiin C; light pink powder; CD (MeOH) [h]214 – 32,679, [h]226 +15,458, [h]240 2678, [h]271 +9110, [h]294 7838; UV (MeOH) kmax (log e) 218 (4.9), 278 (4.5) nm; 1H NMR see Table 3; 13C NMR see Table 2; HR-ESI-MS (negative ion mode): m/z 1348.2446 [MH] (calcd for C57H55O38, 1347.2372); ESI-MS/MS (negative ion mode) see Table 1. D-glucopyranoside

4.3.4. 6-O-(3-(6-acetyl)-glucosyl)-galloyl-1,3-di-O-galloyl-2,4(20 S,30 S,40 S)-chebuloyl-b-D-glucopyranoside (4) Sylvatiin D; light yellow powder; CD (MeOH) [h]218 +56,259, [h]242 13,433, [h]256 5544, [h]269 7857, [h]298 +7452; UV (MeOH) kmax (log e) 220 (4.9), 280 (4.5) nm; 1H NMR see Table 3; 13 C NMR see Table 2; HR-ESI-MS (negative ion mode): m/z 1159.1598 (calcd for C49H43O33, 1159.1687); ESI-MS/MS (negative ion mode) see Table 1. 4.4. Extraction and isolation of reference compounds Reference compounds were previously isolated from G. sylvaticum (geraniin; Tuominen et al., 2013) and Terminalia

11

chebula (chebulinic acid, chebulanin and chebulinic acid, Moilanen et al., 2013). Pentagalloylglucose was synthetized from tannic acid (Hagerman, 2002). The structures of reference compounds are presented in Fig. S4. 4.4.1. 1,2,3,4,6-penta-O-galloyl-b-D-glucopyranoside Pentagalloylglucose; white powder; CD (MeOH) [h]216 31,961, [h]226 +25,820, [h]241 868, [h]273 +32,761, [h]300 19,014; UV (MeOH) kmax (log e) 218 (5.0), 281 (4.6) nm; 1H NMR see Table 3; 13 C NMR see Table 2. 4.4.2. 6-O-galloyl-1,3-di-O-galloyl-2,4-(20 S,30 S,40 S)-chebuloyl-b-Dglucopyranoside Chebulinic acid; white powder; CD (MeOH) [h]218 +66,268, [h]240 20,400, [h]260 1228, [h]273 2845, [h]297 +12,639; UV (MeOH) kmax (log e) 220 (4.9), 280 (4.5) nm. 4.5. RDA test Protein precipitation capacity was measured with the radial diffusion assay with some small changes to the previously described method (Hagerman, 1987). Bovine serum albumin (BSA, SigmaAldrich, St. Louis, USA) was used as a protein in an agarose gel (Agarose type I, Sigma-Aldrich, St. Louis, USA) Petri dish. Compounds were diluted in 40% aq. MeOH and placed in a well in the protein-containing agarose gel. After incubation of 72 h at 30 °C, the diameter of visible rings, that are developed when compound diffuses into the gel and complexes with protein, were measured. The diameter of the ring is dependent of the amount of tannin in the sample and the tannin type and its precipitation capacity. Pentagalloylglucose was used as a standard. Pentagalloylglucose yielded a standard curve with r2 = 0.9713, slope was 29.60 (±1.41) and intercept was 50.07 (±3.73). Three replicates were measured for each sample and standard. The square of the diameter of sample rings was compared with the square of the diameter of pentagalloylglucose standard in the same concentration yielded from the standard curve. 4.6. Extraction for quantification of pigments and petal color comparison The tentative identification of G. sylvaticum petal compounds was presented earlier in Tuominen et al., 2013. Collected petals were air- or freeze-dried and homogenized into fine powder. Ca. 5 mg of fine plant powder were extracted with 700 ll of acetone:water (7:3, v/v) 3 using vortex. Extract was dissolved to 1.5 ml of water and further diluted if necessary. Extracts and the pressed juice sample were filtered through 0.2 lm PTFE filter and analyzed with UPLC-DAD/ESI-MS using the negative ion mode. Four of the main flavonoids were quantified using UV detection at wavelength 349 nm. Quercetin diglycoside was quantified as quercetin 3-(2-galloyl)-glycoside equivalents and kaempferol diglycoside as kaempferol galloylglucoside equivalents. Main anthocyanin and four sylvatiins were quantified using optimized selected reaction monitoring (SRM) methods due to unsufficient resolution in the UV chromatogram. Acetylmalvin was calculated as malvin equivalents. Used SRM methods are presented in the Table 6. 4.7. Co-pigmentation test The second Sephadex LH-20 fraction of petal crude extract eluted with water was dark purple and contained mainly anthocyanins; main compound was acetylmalvin (Tuominen, 2013). This fraction was used as a model sample of petal anthocyanins of G. sylvaticum. A sample soln. of this fraction was prepared

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

12

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx

Table 6 Optimized SRM methods for compound quantification. Compound

Parent ion m/z

Daughter ion m/z

Cone voltage (V)

Collision energy (V)

Sylvatiin A (1) Sylvatiin B (2) Sylvatiin C (3) Sylvatiin D (4) Malvin Acetylmalvin

571.2 495.3 673.3 579.2 671.2 713.2

169 169 169 169 509.2 551.1

30 20 30 30 20 20

25 30 35 25 20 20

by dissolving 10 mg aliquot to the 5 ml of MeOH:water (3:7, v/v). Commercial malvin chloride (malvidin-3,5-diglucoside, Extrasynthese, Genay, France) was used as another model pigment and dissolved to (3:7, v:v) MeOH:water in 1 mM concentration. The co-pigment samples were prepared to the same solvent in 2 mM concentration. Sylvatiin E was used instead of sylvatiin B because there was not enough purified compound left. Buffer solns. (pH 4, 5, 6 citrate and 7, 8 phosphate) were commercial buffer solns. from FF-Chemicals Ab (Haukipudas, Finland) and pure water was used as a reference soln. Solns. were mixed in a well plate: 80 ll of pigment, 90 ll of co-pigment, 20 ll of buffer and 80 ll of water were added. Pigment to co-pigment ratio 1:2 was chosen for study based on concentrations observed in the petal extracts and pressed juice (Table 5). Samples were equilibrated for ca. 1 h. Well plates were shaken for 5 s after addition of solns. and before analysis and then the absorbance at wavelengths 520 and 550 nm was analyzed with a 96-well plate reader (Multiscan Ascent, Thermo Electron Corporation) with three replicate analyses. The average temp. inside the well plate reader was 22.6 °C. Color stability values (Table S1) were calculated from the malvin sample measurements before and after 1 h incubation. The color change in well plates was also photographed and the UV spectra of solns. were measured separately in a 100 ll microcuvette with HALO DB-20 UV–Vis double beam spectrophotometer (Dynamica GmbH, Dietikon, Switzerland). Modified conditions were used to produce clear spectra for the Fig. 5: stronger solutions were used (4 mM for both pigment and co-pigment), the ratio of solns. was 90:80:30:100 respectively, and the spectra were measured as soon as possible after mixing (ca. 1 min) to avoid the fading of samples without co-pigment. Acknowledgements This work was funded by a grant from the Emil Aaltonen Foundation and Turku University Foundation (to A.T.) and the Academy of Finland (Grant no. 251388 to M.K.). Authors want to thank Dr. Jaana Liimatainen for the assistance in CD measurements, Anne Koivuniemi, Johanna Moilanen and Anni Savolainen for the help in the RDA tests and/or the UPLC-MS quantifications. Two anonymous reviewers helped to improve the earlier version of the manuscript. The chemical analyses using the UPLC-MS system were made possible by a Strategic Research Grant of the University of Turku (Ecological Interactions). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2015. 01.005. References Appel, H.M., 1993. Phenolics in ecological interactions: the importance of oxidation. J. Chem. Ecol. 19, 1521–1552. Andersen, Ø.M., Viksund, R.I., Pedersen, A.T., 1995. Malvidin 3-(6-acetylglucoside)5-glucoside and other anthocyanins from flowers of Geranium sylvaticum. Phytochemistry 38, 1513–1517.

Asen, S., Stewart, R.N., Norris, K.H., 1972. Copigmentation of anthocyanins in plant tissues and its effect on color. Phytochemistry 11, 1139–1144. Asikainen, E., Mutikainen, P., 2005. Preferences of pollinators and herbivores in gynodioecious Geranium sylvaticum. Ann. Bot. 95, 879–886. Baranac, J.M., Petranovic´, N.A., Dimitric´-Markovic´, J.M., 1997. Spectrophotometric study of anthocyan copigmentation reactions. 2. Malvin and the nonglycosidized flavone quercetin. J. Agric. Food Chem. 45, 1694–1697. Barbehenn, R.V., Jones, C.P., Karonen, M., Salminen, J.-P., 2006a. Ellagitannins have greater oxidative activities than condensed tannins and galloylglucoses at high pH: potential impact on caterpillars. J. Chem. Ecol. 32, 2253–2267. Barbehenn, R.V., Jones, C.P., Karonen, M., Salminen, J.-P., 2006b. Tannin composition affects the oxidative activities of tree leaves. J. Chem. Ecol. 32, 2235–2251. Brouillard, R., Mazza, G., Saad, Z., Albrecht-Gary, A.M., Cheminat, A., 1989. The copigmentation reaction of anthocyanins: a microprobe for the structural study of aqueous solutions. J. Am. Chem. Soc. 111, 2604–2610. Brouillard, R., Wigand, M.-C., Dangles, O., Cheminant, A., 1991. pH and solvent effects on the copigmentation reaction of malvin with polyphenols, purine and pyrimidine derivatives. J. Chem. Soc. Perkin Trans. 2 (8), 1235–1241. Cai, Y., Lilley, T.H., Haslam, E., 1990. Polyphenol-anthocyanin copigmentation. J. Chem. Soc., Chem. Commun., 380–383 Goto, T., Kondo, T., 1991. Structure and molecular stacking of anthocyanins – flower color variation. Angew. Chem. Int. Ed. Engl. 30, 17–33. Hagerman, A.E., 1987. Radial diffusion method for determining tannin in plant extracts. J. Chem. Ecol. 13, 437–449. Hagerman, A.E., 2002. Pentagalloyl Glucose. In: Tannin Handbook. (accessed by 28.4.2014). Haslam, E., 1998. Anthocyanin copigmentation – fruit and floral pigments. In: Practical polyphenolics. From structure to molecular recognition and physiological action. Cambridge University Press, UK, pp. 262–297. Kawamoto, H., Nakatsubo, F., Murakami, K., 1995. Quantitative determination of tannin and protein in the precipitates by high-performance liquid chromatography. Phytochemistry 40, 1503–1505. Kawamoto, H., Nakatsubo, F., Murakami, K., 1996. Stoichiometric studies of tanninprotein co-precipitation. Phytochemistry 41, 1427–1431. Lightner, D.A., 1994. Determination of absolute configuration by CD. Applications of the octant rule and the exciton chirality rule. In: Purdie, N., Brittain, H.G. (Eds.), Analytical Applications of Circular Dichroism. Elsevier Science B.V., Amsterdam, pp. 131–174. NatureGate, 2014. (accessed 30.4.2014). Nave, F., Petrov, V., Pina, F., Teixeira, N., Mateus, N., de Freitas, V., 2010. Thermodynamic and kinetic properties of a red wine pigment: catechin-(4,8)malvidin-3-O-glucoside. J. Phys. Chem. B 114, 13487–13496. Markham, K.R., Mitchell, K.A., Boase, M.R., 1997. Malvidin-3-O-glucoside-5-O-(6acetylglucoside) and its colour manifestation in ‘Johnson’s blue’ and other ‘blue’ geraniums. Phytochemistry 45, 417–423. Mistry, T.V., Cai, Y., Lilley, T.H., Haslam, E., 1991. Polyphenol interactions. Part 5. 1 Anthocyanin co-pigmentation. J. Chem. Soc. Perkin Trans. 2, 1287– 1296. Moilanen, J., Sinkkonen, J., Salminen, J.-P., 2013. Characterization of bioactive plant ellagitannins by chromatographic, spectroscopic and mass spectrometric methods. Chemoecology 23, 165–179. Nishizawa, M., Yamagishi, T., 1982. Tannins and related compounds. Part 5. Isolation and characterization of polygalloylglucoses from Chinese gallotannin. J. Chem. Soc., Perkin Trans. 1 12, 2963–2968. Okuda, T., Yoshida, T., Hatano, T., Koga, T., Toh, N., Kuriyama, K., 1982. Circular dichroism of hydrolysable tannins – ellagitannins and gallotannins. Tetrahedron Lett. 23, 3937–3940. Pfundstein, B., El Desouky, S.K., Hull, W.E., Haubner, R., Erben, G., Owen, R.W., 2010. Polyphenolic compounds in the fruits of Egyptian medicinal plants (Terminalia bellerica, Terminalia chebula and Terminalia horrida): characterization, quantitation and determination of antioxidant capacities. Phytochemistry 71, 1132–1148. Salminen, J.-P., Ossipov, V., Loponen, J., Haukioja, E., Pihlaja, K., 1999. Characterisation of hydrolysable tannins from leaves of Betula pubescens by high-performance liquid chromatography– mass spectrometry. J. Chromatogr. A 864, 283–291. Salminen, J.-P., Karonen, M., 2011. Chemical ecology of tannins and other phenolics: we need a change in approach. Funct. Ecol. 25, 325–338. Taiz, L., 1992. The plant vacuole. J. Exp. Biol. 172, 113–122.

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005

A. Tuominen et al. / Phytochemistry xxx (2015) xxx–xxx Tanaka, T., Fujisaki, H., Nonaka, G.-I., Nishioka, I., 1992. Tannins and Related compounds. CXVII. Structures, preparation, high-performance liquid chromatography and some reactions of dehydroellagitannin-acetone condensates. Chem. Pharm. Bull. 40, 2937–2944. Tanaka, T., Zhang, H., Jiang, Z.-H., Kouno, I., 1997. Relationship between hydrophobicity and structure of hydrolysable tannins and association of tannins with crude drug constituents in aqueous solution. Chem. Pharm. Bull. 45, 1891–1897. Toivonen, E., Mutikainen, P., 2012. Differential costs of reproduction in females and hermaphrodites in a gynodioecious plant. Ann. Bot. 109, 1159–1164. Tuominen, A., Toivonen, E., Mutikainen, P., Salminen, J.-P., 2013. Defensive strategies in Geranium sylvaticum, Part 1: Organ-specific distribution of water-soluble tannins, flavonoids and phenolic acids. Phytochemistry 95, 394–407.

13

Tuominen, A., 2013. Defensive strategies in Geranium sylvaticum, Part 2: Roles of water-soluble tannins, flavonoids and phenolic acids against natural enemies. Phytochemistry 95, 408–420. Varga, S., Nuortila, C., Kytöviita, M.-M., 2013. Nectar sugar production across floral phases in the gynodioecious protandrous plant Geranium sylvaticum. PLoS One 4, e62575–e62575. Vihakas, M., Pälijärvi, M., Karonen, M., Roininen, H., Salminen, J.-P., 2014. Rapid estimation of the oxidative activities of individual phenolics in crude plant extracts. Phytochemistry 103, 76–84. Yoshida, T., Okuda, T., Koga, T., Toh, N., 1982. Absolute configurations of chebulic, chebulinic and chebulagic acid. Chem. Pharm. Bull. 30, 2655–2658. Yoshida, K., Mori, M., Kondo, T., 2009. Blue flower color development by anthocyanins: from chemical structure to cell physiology. Nat. Prod. Rep. 26, 884–915.

Please cite this article in press as: Tuominen, A., et al. Sylvatiins, acetylglucosylated hydrolysable tannins from the petals of Geranium sylvaticum show copigment effect. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.01.005