Friday, October 21, 2011

Hydrophilic Carboxylic Acids and Iridoid Glycosides in the

Juice of American and European Cranberries (
Vaccinium

macrocarpon
and V. oxycoccos), Lingonberries (V. vitis-idaea),

and Blueberries (
V. myrtillus)

H
EIDI D. JENSEN,†,‡ KAREN A. KROGFELT,CLAUS CORNETT,§

S. H
ONOREÄ HANSEN,§ AND S. BRØGGER CHRISTENSEN*,†

Department of Medicinal Chemistry, Royal Danish School of Pharmacy, Copenhagen, Denmark,

Department of Gastro-Intestinal Infections, Statens Serum Institut, Copenhagen, Denmark, and

Department of Analytical and Pharmaceutical Chemistry, Royal Danish School of Pharmacy,

Copenhagen, Denmark

Analysis of the hydrophilic fraction of cranberry juice by reversed-phase HPLC using an Aqua LUNA

column with diode array or MS detection revealed the presence of quinic acid, malic acid, shikimic

acid, and citric acid. For the first time, two iridoid glucosides were found in the juice. The two iridoid

glucosides were shown to be monotropein and 6,7-dihydromonotropein by MS and NMR spectroscopy.

A fast reversed-phase HPLC method for quantification of the hydrophilic carboxylic acids was

developed and used for analyses of cranberry, lingonberry, and blueberry juices. The level of

hydrophilic carboxylic acids in cranberries was 2.67
-3.57% (w/v), in lingonberries 2.27-3.05%, and

in blueberries 0.35
-0.75%. In lingonberries both iridoid glucosides were present, whereas only

monotropein was present in blueberries.

KEYWORDS: HPLC; American cranberry (Vaccinium macrocarpon); European cranberry (Vaccinium

oxycoccus); lingonberry (Vaccinium vitis-idaea); blueberry (Vaccinium myrtillus); quinic acid; malic acid;

shikimic acid; citric acid; iridoid glucosides; monotropein; 6,7-dihydromonotropein

INTRODUCTION

Approximately 25% of women will have at least one urinary

tract infection in their lifetime caused by infection by bacteria,

especially
Escherichia coli (1). Many of these will have several

infections (
2). Whereas some clinical trials have shown that

cranberry juice prevents urinary tract infections in women (
3,

4
), other reports still question the beneficial effects (5, 6). In

vitro experiments have revealed that cranberry fruit juice

(
Vaccinium macrocarpon Aiton, family Ericaceae) possesses

an antimicrobial (
7) and an antiadhesive effect (8) on E. coli.

Since lack of sensitivity toward common antibiotics increasingly

complicates treatment of infections, antiadhesion therapy might

become an important alternative to the presently used methods.

Some previous studies have related the antiadhesion effects to

the presence of proanthocyanidins (
9), fructose, and macromolecules

(
8) in the juice, whereas other studies have correlated

the effect to the acidificaton of urine caused by the organic acids

(
5). Despite these hypotheses, only very limited attention has

been paid to the hydrophilic fraction, maybe because of

difficulties in performing such analyses. Reversed-phase column

packing materials applicable for separation of very

hydrophilic compounds have enabled development of the fast

method for analyses of the hydrophilic carboxylic acids presented

in this paper. Previous studies have only elucidated the

structures of the very polar compounds by their retention times

(
10, 11). The present study was undertaken in order to confirm

the structures of the polar acids present in cranberry juice and

to identify possible unknown constituents which might contribute

to the biological activity and to the taste of the juice.

Techniques such as HPLC-MS and HPLC-NMR have facilitated

unequivocal structural determination of the detected

compounds. For comparison the study also includes analyses

of the juices of European cranberries (
Vaccinium oxycoccos L.),

lingonberries (
Vaccinium Vitis-idaea L.), and blueberries (Vaccinium

myrtillus
L.).

EXPERIMENTAL PROCEDURES

Chemicals and Materials.
All solvents and reagents were of

analytical grade. Trifluoroacetic acid 99% was from Aldrich, and

acetonitrile and methanol were HPLC-grade. Water was deionized and

filtered through a 0.45
ím poresize Millipore filter (Millipore, Ireland)

before use. Authentic (
-)-quinic acid (1â,3R,4R,5â-tetrahydroxycy-

* Corresponding author. Address: Department of Medicinal Chemistry,

Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen

Ø, Denmark. Tel.:
+45 35306253. Fax: +45 3530 6040. E-mail:

sbc@dfh.dk.

Department of Medicinal Chemistry, Royal Danish School of Pharmacy.

Department of Gastro-Intestinal Infections, Statens Serum Institut.

§
Department of Analytical and Pharmaceutical Chemistry, Royal Danish

School of Pharmacy.

J. Agric. Food Chem.
2002, 50, 6871-6874 6871

10.1021/jf0205110 CCC: $22.00 © 2002 American Chemical Society

Published on Web 10/11/2002

clohexane carboxylic acid) (Aldrich), l-(
-)-malic acid (S-hydroxysuccinic

acid) (Fluka), (
-)-shikimic acid (3R,4S,5R)-3,4,5-trihydroxycyclohexene-

1-carboxylic acid) (Aldrich), and citric acid (2-hydroxy-

1,2,3-propanetricarboxylic acid) (Riedel-de Hae¨n) were used as standards.

Pectinex Ultra SP-L (Novozymes) was used as pectinase. Solidphase

extraction catridges were 500 mg, 3 mL C-18 BondElut (Varian).

RP-18 LiChroprep, 40
-63 ím silylated silica gel (Merck), was used

for preparative removal of the less hydrophilic constituents of the juices.

NMR Apparatus.
The NMR spectra were recorded on a Gemini

2000 spectrometer at 300 and 75 MHz for
1H- and 13C NMR spectra,

respectively. The spectra were recorded in deuterium oxide using

acetonitrile (2.06 ppm) as an internal standard.

HRMS.
The high-resolution mass spectra were recorded on a Q-Tof1

(Micromass) with a 3.6 GHz TDC. Negative ions obtained after electron

spray were detected using a mixture of corn syrup and maltose as

references.

HPLC Apparatus.
The quantitative analyses were performed using

a Water Associates pump Model 510, a Rheodyne 7125 injector valve

with a 20
íL injection loop, and a Shimadzu SPD-10A UV-vis

Detector. Data acquisition and manipulation were performed on a

C-R8A Chromatopac, Shimadzu, Japan. Separation was accomplished

at room temperature on a 150
4.6 mm i.d., 5 ím Aqua LUNA C-18

column (Phenomenex). The preparative isolation of the compounds was

performed on a 150
21.2 mm i.d., 5 ím Aqua LUNA C-18 column

(Phenomenex) and with the same apparatus as for the quantitative

analysis except for a 2 mL loop and a preparative UV detector cell.

HPLC-MS was performed on an 1100 HPLC system equipped with a

diode array and a mass spectrometric detectors (Agilent Technologies,

USA), and HPLC NMR was performed on a Bruker spectrometer

working at 400 MHz using a flow cell.

Berry Samples.
The origins of the berries used in this study are

given in
Table 1. Samples 1, 2, 3, 6, 8, and 9 were purchased in a

grocery store. Sample 4 was a generous gift from Rynkeby A/S,

Denmark. One of the authors collected sample 5 in the fall 2000 in

Bøllemose moor, north of Copenhagen, Denmark, and sample 7 in the

fall 2001 in the Alps near Lausanne, Switzerland. The identities of

samples 5 and 7 were confirmed by Dr. Per Mølgaard, The Royal

Danish School of Pharmacy. All the berries were frozen immediately

after collection or purchase and stored at
-20 °C.

Sample Preparation.
Frozen berries were thawed at 5 °C overnight,

and 1 kg of the berries blended with 0.7 L of deionized water for five

minutes in a Waring Commercial Blendor. The pulp was centrifuged

at 420
g for 15 min in a SIGMA 3 centrifuge. The supernatant was

filtered to give 1 L of juice.

Pectinase Treatment.
The large amount of pectin in blueberries

(samples 8 and 9) necessitated treatment with a pectinase (Pectinex

Ultra) before centrifugation. The pectinase (0.1% v/v) was added to

the pulp, and the pulp was stirred for 60 min at 35
°C. Further sample

preparation was performed as described above.

Quantitative Analysis of the Hydrophilic Carboxylic Acids.
A

BondElut cartridge was activated by washing with 10 mL of acetonitrile
-

water (1:1) and dried by sucking 10 mL of air through the

cartridge. Two portions of 5 mL of the fresh juice were sucked through

the cartridge. The first 5 mL of eluate was discarded and the following

5 mL collected. After filtration through 0.45
ím poresize Whatman

filter, 200
íL of the filtrate was diluted to 5000 íL, and 100 íL of the

diluted sample was injected into the HPLC system using water

containing 0.06% of trifluoroacetic acid as the mobile phase, flow 1.0

mL/min, detection 214 nm. Quantification was based on linear

regression analysis of the ratio between the peak areas in the

chromatograms of the juice and the peak areas of the standards.

Linearity.
Standard solutions of authentic samples of quinic acid,

malic acid, shikimic acid, and citric acid were prepared in mobile phase.

The standard curves were based on five or six concentrations each

analyzed in triplicate. The final concentrations of the acids in the

solutions were as follows: quinic acid, 0.125, 0.167, 0.215, 0.301,

0.378, and 0.501 mg/mL; malic acid, 0.064, 0.128, 0.171, 0.205, and

0.256 mg/mL; shikimic acid, 2.35, 3.10, 4.70, 6.27, and 7.50
íg/mL;

and citric acid, 0.124, 0.165, 0.212, 0.247, 0.297, 0.371, and 0.495

mg/mL. Quality control samples were analyzed before an analysis. Both

standard solutions and samples were stored at 5
°C. Good linearity for

quinic acid and citric acid was achieved between 0.124 and 0.495 mg/

mL with correlation coefficients of 0.9992 and 0.9818, respectively,

for malic acid between 0.064 and 0.256 mg/mL with a correlation

coefficient of 0.9982, and for shikimic acid between 2.4 and 9.4
íg/

mL with a correlation coefficient of 0.9991.

Recovery.
The extraction efficiency (recovery) was determined by

adding authentic standards to the juice and comparing the found

amounts of acids with the amounts in unspiked juice. The recovery

data for quinic acid, malic acid, shikimic acid, and citric acid were

103%, 91%, 97%, and 95%, respectively, with coefficients of variation

between 6 and 8.

Stability Studies.
No changes in the contents of the carboxylic acids

could be observed after freezing and thawing of the juice, as revealed

by comparison of the chromatograms of a freshly prepared juice and a

juice which had been frozen at
-20 °C.

Preparative Isolation of Iridoid Glycosides and Carboxylic Acids.

Frozen berries (1020 g) were thawed at 5
°C, and water (700 mL) was

added. The mixture was blended and the pulp centrifuged at 420
g for

15 min. A 200 mL sample of the supernatant was applicated with a

FMI LAB pump model QD (Fluid Metering, Inc. USA) on 20 g of

RP-18 silylated silica gel in a 300
20 mm column activated with

methanol and washed with deionized water. The column was eluted

with 300 mL water, and the eluates were pooled and evaporated in

vacuo at 40
°C to the volume of the applied juice (200 mL). For the

preparative HPLC, the compounds eluted with a flow of 10 mL/min

were collected and the fractions lyophilized. Detection and mobile phase

were as in the quantitative analysis of the compounds. The
1H and 13C

NMR spectra of quinic acid, malic acid, shikimic acid, citric acid, and

monotropein were superimposable to those of authentic samples and

the expected peaks for (M-1) were found in all the mass spectra.

LC-MS Analysis.
An isocratic elution mode with 0.1% (v/v) aqueous

formic acid as an eluent was used at a flow rate of 0.5 mL/min. The

same HPLC column as for the UV detection was used, and the injection

volume was 50
íL. The mass spectrometric detection was performed

using API-ES in the negative mode.

Molecular Analysis Data.
Quinic acid, shikimic acid, and citric

acid were obtained as a colorless solid. Monotropein (16 mg) was

Table 1.
Sample Number, Origin, and Concentration of Four Organic Acids in the Hydrophilic Fractions of Cranberry, Lingonberry, and Blueberry

Juices (% W/V
± RSD %)

sample
a (4) species origin quinic acid (1) malic acid (2) shikimic acid (3) citric acid

1
V. macrocarpon USAa 1.14 ± 0.01 0.82 ± 0.01 0.041 ± 0.0003 1.20 ± 0.01

2
V. macrocarpon USAa 1.180 ± 0.003 0.80 ± 0.01 0.0293 ± 0.0002 1.14 ± 0.01

3
V. macrocarpon Holland

(Thershellingen)

0.892
± 0.003 0.626 ± 0.004 0.059 ± 0.001 1.10 ± 0.01

4
V. macrocarpon Czech Republic 0.605 ± 0.001 1.052 ± 0.002 0.01092± 0.00004 1.863 ± 0.001

5
V. oxycoccos Denmark 0.55 ± 0.01 0.95 ± 0.01 0.00670 ± 0.00004 1.63± 0.01

6
V. vitis-idaea Sweden 0.88 ± 0.03 tr.b 0.0040 ± 0.0002 1.391 ± 0.003

7
V. vitis-idaea Switzerland 1.530 ± 0.001 tr. 1.52 ± 0.01

8
V. myrtillus Germany tr. tr. tr. 0.75 ± 0.01

9
V. myrtillus Argentina tr. tr. tr. 0.350 ± 0.003

a
The two samples were obtained from different suppliers. b tr., trace amounts.

6872
J. Agric. Food Chem., Vol. 50, No. 23, 2002 Jensen et al.

isolated from 200 mL juice as an amorphous colorless solid, corresponding

to 0.01% w/v in single strength cranberry juice. [
R]25

D
)

-
132.1° (c 0.165, H2O). ìmax (measured with a diode array detector,

which prevented measurement of
) (H2O) 239 nm. 1H NMR (D2O,

300 MHz)
ä 7.47 (1H, d, J3,5 ) 1.0 Hz, H-3), 6.29 (1H, dd, J5,6 ) 2.7

Hz,
J6,7 ) 6.0 Hz, H-6), 5.73 (1H, dd, J5,7 ) 1.5, J6,7 ) 6.0 Hz, H-7),

5.67 (1H, d,
J1,9 ) 1.8 Hz, H-1), 4.82 (1H, d, J1¢,2¢ ) 8.1 Hz, H-1¢),

3.94 (1H, dd,
J6¢a,6¢b ) 12.3, J6¢,5¢ ) 2.4 Hz, H-6¢a), 3.75 (1H, dd, J6¢b,6¢a

)
12.3, J6¢b,5¢ ) 5.7 Hz H-6¢b), 3.72 (1H, d, J10a,10b ) 11.4 Hz, H-10a),

3.66 (1H, d,
J10b,10a ) 11.4, H-10b), 3.52 (1H, ddd, J4¢,5¢ ) 9.9 Hz,

J
6a¢,5¢ ) 2.4 Hz, J6¢b,5¢ ) 5.7 Hz, H-5¢), 3.57-3.60 (1H, m, H-5), 3.51

(1H, t,
J2¢,3¢ ) 9.3 Hz, J3¢,4¢ ) 9.0 Hz, H-3’), 3.40 (1H, t, J3¢,4¢ ) 9.0 Hz,

J
4¢,5¢ ) 9.9 Hz, H-4’), 3.28 (1H, dd, J1¢,2¢ ) 8.1 Hz, J2¢,3¢ ) 9.3 Hz,

H-2’), 2.74 (1H, dd,
J1,9 ) 1.8 Hz, J5,9 ) 8.7 Hz, H-9).13C NMR (D2O,

75.5 MHz)
ä 95.38 (C-1), 153.04 (C-3), 111.53 (C-4), 37.73 (C-5),

133.32 (C-6), 138.70 (C-7), 85.81 (C-8), 44.73 (C-9), 67.34 (C-10),

172.33 (C-11), 99.38 (C-1
¢), 73.61 (C-2¢) 76.60 (C-3¢), 70.51 (C-4¢),

77.29 (C-5
¢), 61.55 (C-6¢). MS API-ES negative [M - 1]- m/z ) 389.0.

6,7-Dihydromonotropein (22 mg) was isolated from 200 mL juice as

an amorphous colorless solid, corresponding to 0.01% w/v in single

strength cranberry juice. [
R]25

D
) -204.6° (c 0.100, H2O). ìmax

(measured with a diode array detector, which prevented measurement

of
) (H2O) 239 nm. 1H NMR (D2O, 300 MHz) ä 7.55 (1H, br s, H-3),

5.54 (1H, d,
J1,9 ) 3.6 Hz, H-1), 4.82 (1H, d, J1¢,2¢ ) 8.4 Hz, H-1¢),

3.92 (1H, br d,
J6¢a,6¢b ) 12.3, H-6¢b), 3.74 (1H, dd, J6¢b,6¢a ) 12.3, J6¢b,5¢

)
5.7 Hz H-6’b), 3.62 (1H, d, J10a,10b ) 17.5 Hz, H-10a), 3.56 (1H, d,

J
10b,10a ) 17.5, H-10b), 3.54-3.48 (1H, m, H-5¢), 3.51 (1H, t, J3¢,4¢ )

9.1 Hz, H-3
¢), 3.41 (1H, t, J3¢,4¢ ) J4¢,5¢ ) 9.1 Hz, H-4¢), (1H, d, J1¢,2¢ )

8.1 Hz, H-1
¢), 3.29 (1H, t, J2¢,3¢ ) 9.1 Hz, J1¢,2¢ ) 8.1 Hz, H-2¢), 2.95

(1H, m, H-5), 2.34 (1H, dd,
J1,9 ) 3.6 Hz, J5,9 ) 9.3 Hz, H-9), 2,08-

1.63 (4H, m, 2H-6, 2H-7).
13C NMR (D2O, 75.5 MHz) ä 95.6 (C-1),

153.4 (C-3),112.4 (C-4), 32.5 (C-5), 30.1 (C-6), 35.7 (C-7), 82.8

(C-8), 45.7 (C-9), 68.2 (C-10), 172.0 (C-11), 99.4 (C-1
¢), 73.3 (C-2¢),

76.3 (C-3
¢), 70.1 (C-4¢), 77.0 (C-5¢), 61.3 (C-6¢). MS API-ES negative

[M
- 1]- m/z ) 391.1 and exact mass ES negative [M - 1]- m/z )

391.1228, cald for C
16H23O11 391.1240.

RESULTS AND DISCUSSION

Chromatographic Data.
Isocratic elution using aqueous

trifluoroacetic acid (0.06%) as an eluent afforded good separation

of a series of carboxylic acids in the most hydrophilic

fraction of the juice (
Figure 1).

Structural Elucidation.
The carboxylic acids, quinic acid

(peak 2), malic acid (peak 3), shikimic acid (peak 4), and citric

acid (peak 5), had previously been stated to be present in the

juice (
10, 11), but no verification of the structures had been

given. HPLC-NMR and HPLC-MS was used in this study to

establish the structures. The above four carboxylic acids were

isolated by preparative HPLC and comparison of their
1H NMR

spectra with those of authentic samples unequivocally established

their structures. In addition to these four carboxylic acids,

two additional peaks at 10.9 and 16.3 min were observed in

the chromatogram. The characteristic signal at 7.5 ppm and the

presence of a glucopyranosyl moiety as revealed by the signal

pattern in the HPLC-
1H NMR spectrum indicated that both of

these compounds were iridoid glucosides. After isolation by

preparative HPLC, the
1H NMR and 13C NMR spectra of the

more hydrophilic iridoid glucoside were found to be superimposable

to the spectra of monotropein (
12). The spectra of the

less polar iridoid glucoside were very similar to those of

monotropein except for the missing signals originating in the

nuclei at the 6,7 double bond. Instead, a complex pattern was

found at 2.08
-1.63 ppm in the 1H NMR spectrum and two

signals at 30.1 and 35.7 ppm in the
13C NMR spectrum. MS

revealed that the molecular weight of the compound was two

units higher than that of monotropein. Consequently the

compound was concluded to be 6,7-dihydromonotropein. The

methyl ester of 6,7-dihydromonotropein (splendoside) has

previously been isolated from
Fouquieria splendens (13), but

this is the first report of natural occurrence of the free acid.

The structures of the compounds isolated from the cranberry

juice are given in
Figure 2. Monotropein is proven to be present

in all the analyzed
Vaccinium species by LC-MS. 6,7-Dihydromonotropein

was proven to be present in cranberries and

lingonberries by LC-MS, but absent in blueberries. A compound

with the
Rf values of monotropein on TLC and PC has

previously been demonstrated in the green parts of
V. oxycoccos

L. (European cranberries), in the stem and fruit of
V. myrtillus

L. (blueberry), and in the leaves and stem of
V. Vitis-ideae L.

(lingonberry) (
14).

Quantification of Quinic, Malic, Shikimic, and Citric Acid.

The amounts of the four carboxylic acids (
1-4) in cranberry,

Figure 1.
Chromatogram of the hydrophilic fraction of cranberry juice

(
Vaccinium macrocarpon). Peak at 2.4 min quinic acid (1), 2.9 min malic

acid (
2), 3.2 min shikimic acid (3), 5.0 min citric acid (4), 10.9 min

monotropein (
5), and 16.3 min 6,7-dihydromonotropein (6). Detection at

240 nm.

Figure 2.
Structures of the isolated acids and iridoid glucosides,

(
-)-quinic acid (1), (-)-shikimic acid (2), L-malic acid (3), citric acid (4),

monotropein (
5), and 6,7-dihydromonotropein (6).

Hydrophilic Acids in Cranberries
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6873

lingonberry, and blueberry juices are given in
Table 1, which

reveals that no major differences in the concentrations of the

acids are found in the different samples of juices. The results

are in agreement with previous finding in juice from
V.

macrocarpon
(10, 11, 14). The analyses were extended to a

series of juices prepared from berries of other
Vaccinium species

such as European cranberries, lingonberries and blueberries

(
Table 1). Preliminary studies revealed that the two iridoid

glucosides do not contribute to the antiadhesive effect of the

juice, but the presence of acids and iridoid glucosides contributes

to the taste of the products. Consequently analyses of the

contents of such compounds are important for making products

with similar tastes. The new analytical protocol facilitates

quantification of the carboxylic acids and has revealed the

presence of iridoid glucosides in cranberry, lingonberry, and

blueberry juices.

ACKNOWLEDGMENT

We thank Carina Jensen, Rynkeby A/S, Denmark for European

cranberries (
V. oxycoccus), Eva Langhoff, NovoZymes for

Pectinase, S. Rosendal Jensen, The Technical University of

Denmark, for a sample of monotropein, students Inge Lise,

Finne´ Nielsen, and Christina Fischer for helping in the

preliminary studies, and Bruker for lending us a flow probe.

Gustav Bojesen (The University of Copenhagen) recorded the

HRMS spectra. The Danish Medical Research Council is

thanked for financial support (grant 9800 989) to K.A.K.

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Received for review May 1, 2002. Revised manuscript received August

22, 2002. Accepted August 22, 2002.

JF0205110

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