Anthocyanins from cell suspension cultures of Daucus carota

Phytochemistry, Vol. 31, No 5, pp. 1593 1601, 1992 Printed in Great B&m.

ANTHOCYANINS

0031-9422/92 $5.00+0.00 (c 1992 Pergamon Press plc

FROM CELL SUSPENSION DAUCUS CAROTA

CULTURES

OF

WERNERE. GLABGEN,VICTORWRAY,* DIETERSTRAcK,tJ~RG W. METZGER~and HANNS U. SEITZ$

Botanisches Institut der Universitlt Tiibingen, Allgemeine Botanik und Pflanzenphysiologie, Auf der Morgenstelle 1, D-7400 Tiibingen, Germany; *Gesellschaft fiir Biotechnologische Forschung (GBF), Mascheroder Weg 1, D-3300 Braunschweig, Germany; 7Institut fiir Pharmazeutische Biologie der Technischen Universitat Braunschweig, Mendelssohnstrasse 1, D-3300 Braunschweig, Germany; SInstitut fiir Organ&he Chemie der Universitat Tiibingen, Auf der Morgenstelle 18, D-7400 Tiibingen, Germany (Receioed in revisedform 11 October 1991)

Key Word

Index-Caucus carotn; Apiaceae; carrot cell suspension cultures; acylated anthocyanins; cyanidin 3-lathyrosidq cyanidin 3-(2”-xylosyl-6”~glucosylgalactoside); Ccoumaroyl, feruloyl, 4-hydroxybenzoyl and sinapoyl derivatives; intramolecular copigmentation.

Abstract-Six anthocyanins were isolated from cell suspension cultures of an Afghan cultivar of Daucus carota by PC or HPLC. The structures of these compounds were elucidated by spectroscopic methods as cyanidin 3-O-lathyroside, cyanidin 3-~-(2”-~-~-D-xylopyranosyl-6”-0_B-D-glucopyranosyl-~-D-galactopyranoside), and the latter acylated with 4-coumaric, ferulic, 4-hydroxybenzoic or sinapic acid. Unusual ‘H NMR chemical shifts and ‘H NOE data indicate an intramolecular copigmentation of the aglycone with these aromatic residues.

INTRODUCTION

Anthocyanins in Daucus carota L. were first investigated in roots of the ‘black carrot’, a cultivated form from India (var. sativa) [I], resulting in the identification of a diglycoside of cyanidin. In a comparison of various tissues of different Daucus species [Z], four different cyanidin glycosides were found in the Indian ‘black carrot’ and in leaves and flowers of the wild carrot (subsp. carota): cyanidin 3-xylosylgalactoside, 3-xylosylglucosylgalactoside and the same triglycoside acylated with ferulic or sinapic acid. Two further compounds, feruloyl and sinapoyl derivatives of cyanidin 3-glucosylgalactoside occurred in the stem of Daucus carota L. subsp. maritimus [2]. Four anthocyanins could be separated from roots of Daucus carota L. subsp. boissierii, a cultivar from Afghanistan, using TLC [3]. In another study, the pigment of the ‘black carrot’ was reported to be malvidin 3-monoglucoside and/or peonidin 3-monoglucoside, but this identification was based only on spectrophotometric data [4]. The reports on anthocyanins found in carrot tissue and cell cultures have been reviewed recently [S]. Apart from one observation of malvidin 3,5-diglucoside [6], only cyanidin has been found to be present as an aglycone. The major pigment from cell suspension cultures has been established to be cyanidin 3-(sinapoylxylosylglucosylgalactoside) [7, 81 and is thus one of the characteristic compounds found in the intact plant. We now report on the isolation of six anthocyanins from carrot suspension cultures and their identification using NMR and ion spray mass spectrometry.

Dedicated to the memory of the late Professor Toshio Goto. #Author to whom correspondence should be addressed.

Acylation of anthocyanins with aromatic acids is considered to be important for colour stability [9-111 by copigmentation and for the uptake of the pigments into the plant vacuole [8, 121. Conclusions regarding the conformation of the molecules in solution from the NMR data are also discussed in this paper. RESULTS AND DISCUSSION

Chromatographic data

PC and TLC with BAW (n-BuOH-HOAc-H,O, 4: 1: 5, upper phase) allowed the separation of four bands, of which the third was dominant. Using 2D-TLC with 1% HCl in the second diniension, six coloured spots with traces of two others were observed (Table 1, A and B). More pigments were detected than previously 181, presumably because of increased anthocyanin accumulation in response to irradiation with UV light [ 131. In parallel, reversed-phase HPLC analyses with two solvent systems (Table 1, a and b) showed six distinct peaks and a few minor ones. These six peaks as well as the six spots on 2DTLC represent the major anthocyanin constituents of the extract from UV-irradiated carrot cell suspension cultures. Peaks 2, 3 and 5 of the HPLC-profile (Fig. 1) corresponded to one band on lD-TLC and PC with BAW. After deacylation using hydrolysis with KOH [14], only peaks 1 and 2 remained. This indicates that at least four acylated compounds must be present in the carrot extracts. The major compound (peak 4) co-eluted with the substance isolated by Harborne et al. [7]. Six anthocyanins could be isolated by repeated PC or prep. HPLC for mass spectrometric analyses; five of these were obtained in sufficient amounts for NMR studies and were also spotted on TLC for determination of R, in solvents C-E (Table 1).

1593

1594

W. E.

GL&?C;ENet al

Table 1. Chromatographic data from ZD-TLC (system A and B), ID-TLC (GE) reversed-phase HPLC (systems a and b)

HPLC: R,min.’ ’ in system

TLC: R, (x 100) in system Compound

A

B

C

D

1

14 21 21 17.5 21 25

32 (22)* (18)* 15 15 16

58

2 3 4 5 6

39 28.5 31 31

and

E

a

b

7

19

15 9.5 13.5 15

29 20 23 25

8.2 8.8 9.8 10.3 11.1 11.6

12.0 14.4 15.5 16.8 19.8 21.5

*Compounds 2 and 3 could not unequivocally be identified on ZD-TLC. For chromatographic conditions see Experimental. Compounds are numbered in order of elution on HPLC (Fig. 1).

I

I

I

1

I

The visible maxima of 36 were shifted towards higher wavelengths compared to the unacylated compounds 1 and 2. This spectral shift has been interpreted to be caused by interaction between the acyl moiety and B-ring hydroxyl groups [7]. Structure

1 236

56

I

I

I

1

IO

20

30

41 Time Imin)

Fig. 1. Elutlon profile of the anthocyanin extract from carrot cell suspension cultures on reversed-phase HPLC (system b). For chromatographic conditions see Experimental.

U V- VIS spectroscopy

UV-VIS spectra were recorded from six compounds using HPLC-Photodiode array detection [15-171 with

the minimum after each peak computed as reference (Table 2). Each spectrum was thus taken in the solvent present at the actual state of the gradient program. The relation of EWJ J&is, maXwas similar for all six peaks and indicated the presence of anthocyanidin 3-glycosides [is, 191. Compounds 4-6 exhibited distinct absorbance peaks in the UV between 310 and 335 nm, pointing to acylation with hydroxycinnamic acids [18]. The intensity of these absorbance peaks indicated monoacylated derivatives corresponding to HPLC-peaks 4 and 6 and monoor di-acylation in compound 5 [19]. Compound 3 showed no absorbance band in the 310-335 nm range, but a shoulder at 269 nm presumably caused by a different acyl residue (see below).

determination

The structures of the six compounds corresponding to peaks 1-6 in Fig. 1 were determined from NMR and/or ion spray mass spectral data. For 1 and 3-6 2D ‘H COSY spectra established the presence of various fragments in the molecule and also the nature of the acyl moieties in %6. The relative configuration and nature of the sugars were deduced from the magnitude of the vicinal and geminal ‘H -‘H coupling constants determined from the 1D ‘H spectra (Table 3). In all cases the presence of cyanidin was apparent from the comparison of the chemical shifts of the aromatic protons with literature data [19]. It should be noted, however, that the shift of H-4 of the aglycone in 3-6 is approximately 0.5 ppm to a higher field of that in 1 and previously reported values for other compounds [19]. The reason for this will be discussed below. To date H-6 and H-8 have not been unambiguously assigned [ 191. In the present case this was possible as the stability of the solution of 4 enabled ‘H NOE difference spectra to be taken. Thus low power irradiation of both H-2’ and H-6’ gave weak negative NOES for H-8. Confirmation of this assignment was afforded by the observation of a long-range five-bond coupling between H-4 and H-8, expected for these proton dispositions (extended zig-zag pathway, [20]), and not between H-4 and H-6. This diagnostic coupling was observed in all compounds and indicated the correctness of the reverse assignments of H-6 and H-8 in 1 (Table 3). C-3 substitution of the aglycone was indicated from the same series of NOE difference spectra by the observation of NOES between H-l of the galactose moiety and H-4 of the aglycone. Sufficient material was available for extensive NMR studies of 4 and 5 to be undertaken. 2D ‘H-detected one-bond 13C- ‘H correlations [21] allowed the direct correlation of the ‘H and 13C shifts (Table 4) of all the protonated carbons in the molecule, while 2D ‘H-deteccorrelations [22] unambiguted multiple-bond 13Cmm1H

Anthocyanins from Daucus carom cultures ously established the inter-residue connections by observation of the three-bond correlations shown Table 5. At the same time unambiguous assignment the quaternary carbons of ring A and ring B of aglycone followed from the observation of correlations H-4, H-6 and H-8, and ring B protons, respectively, in latter spectra.

the in of the to the

1595

The structures of 1, 3 and 6 were now readily established from the tD and 2D ‘H data by comparison with those of 4 and 5. For 2 there was not enough material to obtain a full ‘H spectrum, although the signal to noise in the region 5.2 to 9.0 ppm was sufficient to indicate that no aromatic acyl group was present. Further structural information and confi~ation of the NMR results was

Table 2. Spectral properties of six anthocyanins obtained by HPLC-Photodiode array detection. The minimum after each peak was calculated as reference Compound

A,, VIS

E*40Wn -K?

A,,, acyI

his.

1

520 nm 520 nm 525 urn 532 nm 529 nm 526 nm

2 3 4 5 6

(330 nm) (327 nm) 269 (325) nm 331 nm 330 nm 317 nm

=Y,s, max

mm

0.26 0.22 0.60 (0.22) 0.61 (O&3-1.55)* 0.75

0.30 0.30 0.35 0.30 0.32 0.33

*The intensity of the acyl Peak of S varied in different samples probably due to the calculation

of the reference.

Table Chemical

1

3 4 5 6

1 3 4 5 6

______-___-__._____________

_____.__

Gal

____-______

_ _.____

H-6

H-8

H-2

H-S

H-6’

H-l

H-2

H-3

H-4

9.017 8.574 8.533 8.508 8.574

6.723 6.648 6.681* 6.678 6.698

6.964 6.899 6.550* 6.533 6.626

8.124 8.068 7.968 7.929 7.988

7.069 7.093 7.061 7.071 7.114

8.328 8.269 8.180 8.117 8.119

5.431 5.280 5.308 5.290 5.315

4.253 4.22 4.324 4.313 4.249

3.94 2 4.234 4.195 4.172

3.96 - - 3.85 3.988 3.997 3.989

H-5 ---3.90 4.34 4.536 4.503 4.476

Gal ____ ____

H-l

H-6B

H-l

4.10---4.14 4.274 4.272 4.238

4.04 3.86 3.788 3.780 3.799

4.311 4.470 4.513 4.516 4.500

H-2

4.744 3.125 4.124 3.26 4.888 3.244 4.769 3.273 4.707 3.290 --------------------------~

Glc____________________________

_-__*___________---_-------_---

H-6A

_*____ ___-______

Wf

CD,OD/DCl

H-4

________

Cpd

l-+5$in

shifts: --~--~_-__~_..~_~Agl

Cpd 1 3 4 5 6

3. ‘H NMR data for anthocyanina

Xyf_______ H-3

3.23 3.42 -3.50 * 3.49 3.46

H-4

H-3 3.37-------3.19 $ -3.50 N 3.49 3.47

# 3.763 3.763 3.743

-_--___-_-___________ H-4

3.338 3.34 3.376 3&l------3.32 * 3.37

H-2

H-5A

H-5B

3.681 3.074 3.64 2.895 3.653 3.172 -3.57 2.963 3.533 2.852 3.4s Acy__--__-__-_______________________

3.434 3.46 3.287

H-2

H-3

H-5

H-6

H-7

H-8

H-10

H-11

7.814 6.246 6.519 6.997

6.457 6.428

6.457 6.388 6.428

7.814 6.246 6.587 6.997

7.356 7.349 7.406

6.233 6.181 6.183

3.495 3.569

3.495

H-5

H&A

H-6B

3.26 3.54 -3.50 -3.49 3.48

3.902 5.14 5.394 5.342 5.279

3.681 4.32 4.165 4.158 4.164

W. E. GLAOGENet al.

1596

Table Coupling

contants:+ _ _ _ _ _ - _ _ Agl

Cpd 1 3 4 5 6

(6-8) 2.0 1.5 1.8 broad 1.9

________

(2’~6’) 2.3 2.0 2.3 2.0 2.3

(S-6’) 8.7 8.4 8.7 8.7 8.7

-____- _-. ,_-_- __Cpd

(l-2)

1

7.7 7.1 7.2

IlO

6.9

no no

3 4 5 6

-7

_____

_____________________~_~__~Gal (l-2) 7.6 7.4 7.5 1.3 7.4 _____ Glc

(2-3)

(4-5)

_- __-_- __

Xyl______

Cpd

(l-2)

(2-3)

(4-5A)

1 3 4 5 6

7.6 7.6 7.8 7.6 7.6

9.1

5.7

9.0 nfo 9.0

9.0 nfo no

3. (Continued)

5.3 nfo 5.4

__________________________

(2-3) 9.4

(3-4)

(4-5)

(5-6A)

(5-6B)

(6A-6B)

9.2 9 9.2

3.3 3 3.3


9.6 9.4 9.6

1.6 small small

13.0 12.6 12.9

._________________-_------(5-6A)

(5-6B)

2.3

5.7

no no

no no

12.0 -12 12.0 12.0

IlO

no

ll0

_- _-____-_-__-_--__ (4-SB) 10.4 - 10.5 10.9 nfo 10.5

(6A-6B)

---_-_-_______Acy

(SA-5B) 11.5 v 11.5 11.5 nfo 11.5

(2-6)

_________________

(5-6)

(7-8)

8.1

15.9 16.0 16.0

8.5 (2-3) +(2-5) 1.7

8.6 (2--3) +(2-5)

*Assignment from the observation of weak NOES between H-2’ and H-8, and H-6’ and H-8 of the aglycone, and from the longrange coupling between H-4 and H-8 observed in the high-field COSY spectra. tno =not observable due to signal overlap, nfo = not first order. $Compounds 3 and 2 occurred as a 2: 1 mixture, together with smaller amounts of other unidentified compounds. Hence only a partial assignment was possible from the 2D COSY spectrum. $The residue abbreviations are Agl = aglycone, Gal = galactose, Glc =glucose, Xyl = xylose and Acy = acyl residue.

afforded by ion spray mass spectrometry. Ion spray, i.e. pneumatically assisted electrospray [23], is a soft ionization method operating at ambient temperature and atmospheric pressure. The ion spray mass spectra of the purified carrot anthocyanins mainly exhibited the molecular peaks [M]’ as the flavylium cations of the intact compounds and the aglycone fragments, thus enabling the identification of derivatives of certain anthocyanidins. An ethyl acetate-washed extract from carrot cell cultures that was directly supplied into the ion source showed a mass spectrum with distinct signals at m/z 949 and 919, representing the molecular masses of the major anthocyanin constituents. The mass spectra of all the purified compounds l-6 contained a fragment with m/z 287 indicating cyanidin as the common aglycone. The experimentally determined molecular masses were in good agreement with the calculated monoisotopic masses and corresponded to the peaks in the HPLC (Fig. l), as given in Table 6. All six compounds contain cyanidin as the aglycone. Five of them are also identical in their glycosidic component (Fig. 2). The major pigment (peak 4), co-eluting with the substance described by Harborne [7, 241 on HPLC and TLC (data not shown), is cyanidin 3-0-[Z”-O-pD-xylopyranosyl-6”-O-(6’V-O-E-sinapoyl-~-D-glucopyranosyl)-P-D-galactopyranoside] with a branched triglycoside, as has recently been confirmed by methylation

analysis and GLC (GkBgen et al., unpublished results). Compound 5 was identified as the same branched cyanidin triglycoside acylated with ferulic acid at C-6 of the glucose while 1 contains the triglycoside without an acyl function. Compound 2 is cyanidin 3-Q”-xylosylgalactoside) or 3-lathyroside. These four compounds have been reported to be present in plants of Caucus carota L. subsp. sativa and subsp. carota [2]. Thus, the suspension culture used in this study reflects the anthocyanin composition of the intact plant. Compound 6 was identified as 4-coumaroyltriglycoside which has been reported from Apium graveolens L. stem [2]. Compound 3 contains 4-hydroxybenzoic acid as the acyl group, an aromatic acid that is less common in anthocyanins [e.g. 25-281 and reported in the Apiaceae for the first time. From the detection of a molecular ion of m/z 905 cooccurring with a cyanidin fragment in ion spray mass spectra of the extracts, it can be concluded that a cyanidin triglycoside acylated with caffeic acid might also be present in the carrot suspension cultures. intramolecular copigmentation

The first indication that some form of inter- or intramolecular interaction was present in the acylated compounds S-6 was the observation of a high field shift of H-4 of the aglycone compared to 1 and literature data for

Anthocyanins from Daucus caroOtacultures

’

a

w

m

1597

W. E. GL~~BGENet al.

1598

cyanidin compounds [19]; through-bond effects from the remote acyl substituent were clearly impossible. The possibility of intermolecular stacking could be disregarded as the ‘H shifts of 4 showed no significant changes upon an eight-fold dilution of the compound (Table 7). In particular the shifts of the aglycone and aromatic acyl substituent remained constant to kO.03 ppm over this concentration range. Consequently since 4 was stable in solution ‘H NOE difference spectra were taken for many of the protons that showed well resolved signals. The results are shown in Table 8, where the NOE interactions have been classified as weak or strong according to visual estimation of the spectra. The largest long-range NOE was observed between H-l (Gal) and H-7 (Acy) which was of the same magnitude as those of the intra-residue NOES from H-l (Gal) to H-2 (Gal), to H-3 (Gal) and to H-5 (Gal). Significantly there were many much weaker, but readily detectable, NOES between the acyl substituent and the cyanidin system. Such interactions are a clear indication that the acyl substituent and aglycone of the same molecule are in close proximity. The observation of weak NOES between H-8 and both H-2’ and H-6’ implies relatively free rotation of the aglycone ring B and, as H-6 showed no detectable NOES to any other proton in the system, the acyl group ring system must be in the vicinity of the pyrylium ring of the flavylium ion oriented in such a way that H-7 of the acyl group comes into close proximity with H-l of the galactose moiety. A parallel arrangement of the two ring systems would account for the shielding effect observed for H-4 caused by the diamagnetic anisotropy of the acyl aromatic system. Such a stacking arrangement has been previously invoked to explain the stability of the chromophores of polyacylated anthocyanins [l 1,291. The present paper, however, is the first report of direct experimental evidence of intra-

molecular copigmentation in monoacylated anthocyanins. Although this phenomenon is expected to be of general occurrence the magnitude of the interaction will critically depend on the structure of the sugar groupings between the two interacting aromatic systems. From our observations the unusual high field shift of H-4 is a useful indicator of the stacking while more direct information can be obtained by the measurement of the weak longrange NOES. The sequence of the acylated compounds on HPLC (Table 1) is unusual in that the hydroxycinnamoyl derivatives elute in the order sinapoyl-, feruloyl- and 4-coumaroyl-triglycoside of cyanidin in both reversedphase HPLC systems. Normally 0-methylation is expected to increase the elution times on RP-HPLC [19] as is the case with free sinapic, ferulic and 4-coumaric acid [30, 311. This observation must be ascribed to the presence of intramolecular copigmentation and is perhaps the first observation of the influence of such properties on chromatographic behaviour. The spectral shifts mentioned above in the visible peaks of the absorption spectra of the acylated pigments are further experimental evidence for copigmentation in carrot anthocyanins. EXPERIMENTAL Cell cultures. Anthocyanin-containing cell suspension cultures of an Afghan cultivar of Daucus carota L. were derived from callus cultures [32, 333 and maintained as previously described [34]. Cells were irradiated with UV light (315420 nm) 7 days after inoculation to increase anthocyanin accumulation Cl33 and harvested after 14 days of cultivation.

Table 5. Inter-residue three bond 13C-lH correlations observed in the 2D ‘H-detected multiple-bond multiple-quantum coherence

spectra

of 4 and 5*

Proton correlated with carbon H-l (Gal) H-2 (Gal) H-6A (Gal) H-6B (Gal)

Proton correlated with carbon

C-3 (Agl) C-l (Xyl) C-l (Glc)

H-l (Glc) H-6A (Glc) H-6B (Glc)

C-l (Glc)

H-l (Xyl)

C-6 C-9 C-9 C-2

(Gal) (Acy) (Acy) (Gal)

l,R=H

3, R = 4-Hydroxybenzoyl 4,

R * S~nopoyl

5,

R - Feruloyl

6,

R = 4- Courmroyl

*The residue abbreviations are Agl =aglycone, Gal = galactose, Glc = glucose, Xyl = xylose and Acy = acyl residue.

Table

6. Identification of the six major anthocyanins in extracts from carrot cultures and molecular masses as obtained from ion spray MS

Compound

Identified

1 2 3 4 5 6

3-(2”-XyM”-Glc-Gal) 3-(2”-Xyl-Gal) 3-[2”-Xyl-6”-(4-hydroxybenzoyl)Glc-Gal] 3-(2”-Xyl-6”-sinapoylGlc-Gal) 3-(2”-Xyl-6”-feruloylGlc-Gal) 3-[2”-Xyl-6”-(4-coumaroyl)Glc-Gal]

as cyamdin

denvative

[Ml’ 743 581 863 949 919 889

cell

8.541 8.545

C

3.763

3.760 3.758

4.513

4.511 4.512

b

C

7.981 7.989

7.938 7.968

H-2

5.396 5.397

5.394

5.389

H-6A

4.168 4.165

4.165

4.166

H-6B

8.183 8,184

8.174 8.180

H-6

4.873 nm.

4.888

4.878

H-l

3.345 3.234

3.244

3.241

H-2

...~~_~~~~~~~..~____‘xyf

7.066 7.070

7.048 7.061

H-5

3.298 3.278

3.287

3.288

H-4

5.314 5.317

5.296 5.308

H-l

4,235 4.239

4.232 4.234

H-3

3.653 3.653

3.653

3.653

H-5A

3.169 3.168

3.172

3.177

H-5B

--____*______________

4.321 4.320

4.325 4.324

H-2

4.532 4.531

4.539 4.536

H-5

6.252 6.255

6.246

6.233

H-2/H-6

7.363 7.366

7.356

7.341

H-7

----_-_--~-~~~~____Acy

3.987 3.985

3.991 3.998

H-4

4.271 4.269

4.262 4.274

H-6A

3.787 3.785

3.793 3.788

H-6B

6.238 6.239

6.233

6.223

H-8

3.499 3.503

3.495

3.485

H-IO/H-II

_---_______________

~~~~~~---~~~~~~----~~~~~~~__~~~~~~~~a~----------~-~--------~~~~~~~~___

n.m. = Not measured as resonance is under residual water signal. a) 10 mg, b) 5 mg, c) 2.5 mg and d) 1.25 mg per 0.6 ml solvent. Residue abbreviations are the same as those given in Table 3.

3.768

4.522

a

H-4

H-l

Cpd

d

6.555 6.558

6.538 6.550

H-8

________________G,c_________________

6.678 6.674

8.514 8.533

a b

d

6.685 6.681

H-4

Cpd

H-6

Chemical shifts: ___________________________Apl-____---____________________

Table 7. ‘H chemical shifts of those signals of anthocyanin 4 which could be accurately measured in CD,OD/DCl at different concentrations

a 6 8 5 ;a

;

z 3 Q 8

E z B e. e

W. E. GLALXEN

1600 Table

Proton

8. Qualitative

negative

NOES

et al.

observed by ‘H difference CD,OD/DCl Protons Strong

irradiated

showing

spectroscopy

for 4* in

NOES Weak

H-7 (Acy), H-IO/H-11 (Acy) H-2/H-6 (Acy) H-4 (Ad), H-8 (Agl) H-2’ (Agl), H-l (Gal) + H-8 (Acy) H-4 (Agl), H-l (Gal) H-7 (Acy) H-2/H-6 (Acy) ______-_________________________________~~~~---~~~~~~~--~~~~~~_---~ H-7 (Acy), H-l (Glc) H-l (Gal) H-4 (Agl) H-6 (Agl) No NOES H-Z’(AgI), H#(Agl) H-8 (Agl) H-S(Agl) H-6’(Agl) H-8 (Agl), H-2 (Gal) H-5’ (Agl) H-6’ (Agl) ______--________--______________________~~~~---~~~~~~~--------~---H-l (Gal)

H-4 (Agl), H-3 (Gal), H-5 (Gal), H-7 (Acy) -_____---_______-_____________________________~._______~~~__~____~~~~

H-l (Xyl)

H-2 (Gal), H-5B (xyl)

*The residue abbreviations are: = xylose and Acy =acyI residue. *

Agl= aglycone,

Isolation ojanthocyanins. Cells were extracted by grinding in a mortar with MeOH-HOAoH,O (50:8:42, MAW). The filtered extract was used for determination of the anthocyanin content by measuring Es,, nm and calculating with log E= 4.48 for cyanidin 3-galactoside [18] (anthocyanin content reached about 5 mgg-‘fr. wt in irradiated cell suspensions). This MAWextract was infiltrated with N,, coned in a rotary evaporator at 35” and washed 4 times with EtOAc. The aq. phase was evapd to dryness and the residue redissolved in a min. vol. of MAW. Four bands were obtained from this crude extract by overrunning PC in the descending mode on Whatman 3MM-paper with nBuOH-HOAc-H,O (4: 1: 5, upper phase, BAW) for 40-48 hr or by prep. HPLC. The bands from the PC were eluted with MAW and purified by repeated PC with 15% HOAc and BAW, and during this process the second band was further separated into three compounds. The progress of the purification was monitored by HPLC with system b (see below). Preparattue HPLC. Anthocyanin extract (l-2 ml) was injected via a rotary valve with a 2-ml loop into a silica Crs column (10 pm, 300 x 40 mm i.d.) and separation was performed at a flow rate of 20 ml min-’ with a linear gradient ranging from 10% to 45% solvent B in (A + B) within 100 min. Solvent A was 10% HCO,H in H,O, solvent B 1% HCO,H and 19% H,O in MeOH. Detection wavelengths were 530 and 280 nm. HPLC-photodiode array detection. A HP 1090 liquid chromatograph equipped with a diode array-detector (Hewlett Packard, Bad Homburg, F.R.G.) was used. Washed extract (20 ~1) was injected into a Hypersil ODS column (3 mxr, 70 x 4.6 mm i.d.), and elution was performed at a flow rate of lmlmin-’ with a linear gradient of 15-60% solvent B in 20 min. Solvent A was 1.5% H,PO, in H,O, solvent B contained 1.5% H,PO,, 20% HOAc and 25% CH,CN in H,O. Detection was performed at 530 and 320 nm at 0.05 absorbance units full scale. This system is referred to as system a in Table 1. Analyttcal HPLC (system b). Another RP-HPLC-separation was achieved on a Hypersil ODS column (5 pm, 250 x 5 mm i.d.) with a two-step linear gradient of 10% HCO,H in H,O (solvent A) and 10% HCO,H in MeOH (solvent B): during O-14 min solvent B increasing from 13% to 16%, and during 14-30 min from 16% to 18% at a flow rate of 1.5 mlmin-i followed by

Gal = galactose,

Glc = glucose,

Xyl

washing of the column with 99% solvent B. Injection volume was 10 4 (rotary valve) and absorbance was detected at 530 nm (Fig 1). TLC. Systems A-D were run on O.l-mm cellulose plates (20 x 20 cm). Solvents: A=BAW, B= 1% HCI in H,O, C =HOAc-HCl-H,O (15:3:82), D=n-BuOH-2M HCl (l:l, BuHCl). System E was developed on 0.25-mm silica gel (20 x 20 cm) with EtOAc-HCO,H-Ha0 (70: 15: 15) as solvent. 2DTLC was performed on cellulose with BAW and 1% HCI. Deacylation with KOH. Carried out according to ref. Cl43 for 4 hr at room temp. in the dark before adding pre-washed Amberlite IR-120 resin (H+-form, Fluka, Buchs, Switzerland) for acidification. The liberated aromatic acids were extracted with Et,O. NMR spectroscopy. ‘H (600.13 MHz) and i3C (150.90 MHz) NMR spectra were recorded at 300 K on Bruker AM 600 NMR spectrometer locked to the major deuterium resonance of the solvent, CD,OD containing a trace of DCl. All 1D and 2D (COSY-45, ‘H-detected one-bond and multiple-bond t3C multiple-quantum coherence spectra) data were recorded, under the conditions described previously [35], using the standard Bruker software package. ‘HNOE difference spectra were recorded using multiplet irradiation with a relaxation delay between data acquisitions of 8 sec. Data manipulation of the 2D spectra were performed on a Bruker Aspect X32 data station. Chemical shifts are reported in ppm relative to tetramethylsilane (TMS) and coupling contants in Hz. Ion spray mass spectrometry. Ion spray mass spectra were recorded on a Sciex API III triple quadrupole mass spectrometer with 2400-Da mass range equipped with an ion spray ion source (Sciex, Toronto, Canada) [23]. Purified anthocyanin samples and anthocyanin extracts dissolved in MAW were introduced directly into the ion spray source at a constant flow rate of 5 ~1 min- ’ with a microhtre syringe using a medicinal infusion pump (Harvard apparatus, U.S.A.). Final concentrations were about 0.1-l mM, total sample consumption was in the nanomolar range. Acknowledgement’swe are grateful to C. Kakoschke (GBF), B. Jaschok (GBF), S. K&k and R. Hofmann for technical

Anthocyanins from Daucus carota cultures assistance. The help of Dr M. Bokem and S. Heuer (Technische Universitlt Braunschweig) is also gratefully acknowledged. W.E.G. was supported by a fellowship (LGFG) of the Land Baden-Wiirttemberg and thanks Prof. J. B. Harhome and J. Greenham for their help in anthocyanin purification during a visit to Reading. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (Se 229/12-3 and SFB 323). REFERENCES

1. Krishnamoorthy, V. and Seshadri, T. R. (1962) J. Sci. Ind. Res. (India) 21B, 591. 2. Harborne, J. B. (1976) Biochem. System. Ecol. 4, 31. 3. Madzharova, D. and Bubarova, M. (1984) Genet. Sel. 17,79. 4. Canbas, A. (1985) Doga Bilim Derg., Seri 02, 9, 394. 5. Seitz, H. U. and Hinderer, W. (1988) in Cell Culture and Somatic Cell Genetics of Plants, Vol. 5; Phytochemicals in Plant Cell Cultures; Anthocyanins (Constabel, F. and Vasil, I. K., eds), pp. 49-76. Academic Press, San Diego. 6. Ibrahim, R. K., Thakur, M. L. and Permanand, B. (1971) Lloydia 34, 175. 7. Harbome, J. B., Mayer, A. M. and Bar-Nun, N. (1983) 2. Naturjbrsch. 3&, 1055. 8. Hopp, W. and !.?eitz,H. U. (1987) Planta 170, 74. 9. Brouillard, R. (1988) in The Flauonoids: Advances in Research since 1980; Flaoonoids and Flower Colow (Harborne, J. B., ed.), pp. 525-538. Chapman & Hall, London. 10. Goto, T., Tamura, H., Kawai, T., Hoshino, T., Harada, N. and Kondo, T. (1986) Ann. N.Y. Acad. Sci. 471, 155. 11. Goto, T. and Kondo, T. (1991) Angew. Chem. 103, Int. Ed. 30, 17. 12. Matern, U., Reichenbach, C. and Heller, W. (1986) Planta 167, 183. 13. Gleitz, J. and Seitz, H. U. (1989) Planta 179, 323. 14. Albach, R. F., Kepner, R. E. and Webb, A. D. (1965) J. Food Sci. 30, 69. 15. Andersen, 0. M. (1985) J. Food Sci. So, 1230. 16. Hebrero, E., Santos-Buelga, C. and Rivas-Gonzalo, J. C. (1988) Am. J. Enol. Vi’itic.39, 227.

Note added in proof

During preparation of this manuscript Yoshida and coworkers published the evidence. for a copigmentation of a monoacylated anthocyanin from Dioscorea alata (Tetrahedron Letters 32, 5579, 1991).

1601

17. Hong, V. and Wrolstad, R. E. (1990) J. Agric. Food Chem. 38, 708. 18. Harborne, J. B. (1967) Comparative Biochemistry of the Flavonoids. Academic Press, London. 19. Strack, D. and Wray, V. (1989) in Methods in Plant Biochemistry, Vol. 1; Plant Phenolics; Anthocyanins (Harborne, J. B., ed.), pp. 325-356. Academic Press, London. 20. Jackman, L. M. and Sternhell, S. (1969) Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry, 2nd edn. Pergamon Press, Oxford.

21. Cavanaah. J., Hunter. C. A.. Jones, D. N. M., Keller. 3. and Sanders, 51i. M. (1988) Magn. Res. Chem. 26, 867. 22. Summers, M. F., Marzilli, L. G. and Bax, A. (1986) J. Am. Chem. Sot. 108,4285. 23. Bruins, A. P., Covey, T. R. and Henion, J. D. (1987) Analyt. Chem. 59, 2642. 24. Harborne, J. B. and Grayer, R. J. (1988) in 7’he Flauonoids: Advances in Research since 1980; The Anthocyanins (Harborne, J. B., ed.), pp. l-20. Chapman & Hall, London. 25. Willstltter, R. and Mieg, W. (1915) JustuYLiebigs Ann. Chem. 408, 61. 26. Anen, S., Stewart, R. N. and Norris, K. H. (1979) Phytochemistry 1% 1251. 27. Nozue, M., Kawai, J. and Yoahitama, K. (1986) J. Plant

Physiol. 129, 81. 28. Terahara, N., Toki, K., Saito, N., Honda, T., Isono, T., Furumoto, H. and Kontani, Y. (1990) J. Chem. Sot., Perkin Trans. I 3327. 29. Brouillard, R. (1983) Phytochemistry 22, 1311. 30. Strack, D., Akavia, N. and Reznik, H. (1980) Z. Naturforsch. 3sc, 533.

31. Schrq A. W., Jonsson, L. M. V. and de Vlaming, P. (1983) Z. Naturforsch. 3&z, 342. 32. Alfermann, W. and Reinhard, E. (1971) Experientia 27, 353. 33. Schmitz, M. and Seitz, H. U. (1972) Z. Pjbmzenphysiol. 68, 259. 34. No&, W., Langebartels, C. and Seitz, H. U. (1980) Planta 149, 283. 35. Schwind, P., Wray, V. and Nahrstedt, A. (1990) Phyto-

chemistry 29, 1903.