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Natural Product Research

ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20

Antioxidant oligomeric proanthocyanidins from

Cistus salvifolius

Fadi Qa’dan , Frank Petereit , Kenza Mansoor & Adolf Nahrstedt

To cite this article: Fadi Qa’dan , Frank Petereit , Kenza Mansoor & Adolf Nahrstedt (2006)

Antioxidant oligomeric proanthocyanidins from Cistus salvifolius , Natural Product Research,

20:13, 1216-1224, DOI: 10.1080/14786410600899225

To link to this article: http://dx.doi.org/10.1080/14786410600899225

Published online: 01 Dec 2006.

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Natural Product Research

, Vol. 20, No. 13, November 2006, 1216–1224

Antioxidant oligomeric proanthocyanidins from

Cistus salvifolius

FADI QA’DAN*y, FRANK PETEREITz,

KENZA MANSOORx and ADOLF NAHRSTEDTz

y

Faculty of Pharmacy, The University of Petra, PO Box 961343, Amman, Jordan

z

Institut fuer Pharmazeutische Biologie und Phytochemie, Hittorfstrasse 56,

48149 Muenster, Germany

x

Faculty of Pharmacy and Medical Technology,

Al Ahliyya Amman University, Amman, Jordan

(Received 31 March 2005; in final form 15 December 2005)

The purified proanthocyanidin oligomers of Cistus salvifolius herb extract accounted for 78%
of the total proanthocyanidins and 73% of the total antioxidant activity of this extract.
To elucidate the structure of the oligomer, it was depolymerized by acid catalysis in the presence
of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were
determined on the basis of 1D- and reverse 2D-NMR (HSQC, HMBC) experiments of their
peracetylated derivatives, MALDI-TOF-MS and CD spectroscopy. These observations
resulting from the degradation with phloroglucinol were confirmed by

13

C NMR spectroscopy

of the oligomer. The mean molecular weight of the higher oligomeric fraction was estimated to
be 5–6 flavan-3-ol-units.

Keywords: Cistus salvifolius

; Antioxidant oligomeric proanthocyanidins; Degradation

1. Introduction

Cistus salvifolius

L. (Cistaceae), a shrub widely distributed in the Mediterranean

area [1], is traditionally used in Jordan for the treatment of gout [2]. Recent research in
Turkey shows that, of the seven plants used as folk remedies for ulcers, the one with the
greatest efficacy was C. salvifolius [3].

Several flavan-3-ols and dimeric prodelphinidins were isolated from the air-dried

herb material of C. salvifolius and were characterized [4].

Among the flavan-3-ols, catechin, gallocatechin, epicatechin, epigallocatechin,

epicatechin-3-O-gallate,

epigallocatechin-3-O-gallate

and

epigallocatechin-3-O-

(4-hydroxybenzoate) were isolated. The presence of the dimeric prodelphinidins such
as epigallocatechin-(4
! 8)-epigallocatechin, epigallocatechin-3-O-gallate-(4
! 8)-
epigallocatechin and epigallocatechin-(4
! 6)-epigallocatechin-3-O-gallate were also
reported. However, little is known about the structural variation of the higher

*Corresponding author. Fax: [phone]. Email: [e-mail]

Natural Product Research

ISSN 1478-6419 print/ISSN 1029-2349 online

ß 2006 Taylor & Francis

http://www.tandf.co.uk/journals

DOI: 10.1080/14786410600899225

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oligomeric proanthocyanidin fraction. This knowledge is of great importance for better
understanding of the chemical structure of proanthocyanidins in relation to their role
in the traditional use of Cistus sp. as anti-inflammatory agents [5] and for the
determination of the contribution of the oligomeric proanthocyanidin fraction to
the total antioxidant activity of the crude extract.

The present investigation deals with the isolation, physical characterization and

degradation of an oligomeric proanthocyanidin fraction from C. salvifolius with
phloroglucinol and the identification of the degradation products.

2. Results and discussion

The defatted crude acetone–water (7 : 3) extract of C. salvifolius herb was repeatedly
extracted with ethyl acetate to remove the flavan-3-ols and the lower oligomeric
proanthocyanidins. The remaining water-soluble residue was applied to an LH-20
Sephadex column to remove carbohydrates from higher oligomeric condensed
tannins [6] by methanol–water (1 : 1) eluted solvent.

However, the higher oligomeric fraction was eluted with acetone–water (7 : 3).

To distinguish which compounds are relevant for the antioxidant activity of
C. salvifolius

herb extract, the ethyl acetate fraction (EAF), the methanol–water

fraction (MWF) and the acetone–water eluted fraction (AWF) were investigated by
ESR spectroscopy (table 1). The antioxidant activity of the three fractions (EAF,
MWF, AWF) was expressed as its ability to reduce a synthetic free radical species using
Fremy’s salt. The EAF contains mainly flavonoids, flavan-3-ols, dimeric proanthocya-
nidins and other phenolic compounds [4].

By HPLC and TLC investigations, it was possible to demonstrate that the MWF

contains mainly carbohydrates, dihydroflavonol glycosides, trimeric and part of the
tetrameric proanthocyanidins [7].

Because of the excellent radical scavenging properties of these compounds,

the EAF and the MWF showed relatively high antioxidant properties (table 1).
However, 73% of the total antioxidant activity of C. salvifolius crude extract was
recovered in the AWF. Furthermore, the highest proanthocyanidin concentration was
measured in the AWF. Thus, our objective was to obtain detailed structural
information on this oligomeric proanthocyanidin fraction.

The purified oligomer fraction (Acetone Water Fraction; obtained s. Exp) showed an

optical rotation of þ77

(c 0.1, MeOH) which corresponds to a molar proportion of

subunits with a relative 2,3-cis stereochemistry of 83% [8]. By integration of the signals

Table 1.

Antioxidant activity and proanthocyanidin content of the fractions isolated from Cistus salvifolius

herb crude extract.

Fraction

Proanthocyanidin

concentration

(mg cyanidin L



1

fraction)

Antioxidant activity

(mmol Fremy’s salt

reduced L



1

fraction)

Percentage of

antioxidant activity (%)

Ethyl acetate (EAF)

465  16

a

9.7  0.1

9.0

Methanol–H

2

O (MWF)

562  21

19.2  0.4

18.0

Acetone–H

2

O (AWF)

3720  63

78  1.5

73.0

a

Data are mean  SD obtained from three different assays.

Antioxidant oligomeric proanthocyanidins from

C. salvifolius

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close to  77 ppm and  84 ppm by

13

C NMR spectroscopy (solvent: MeOH-d

4

, 99 MHz)

of the oligomer (AWF), a ca 4 : 1 ratio was obtained for cis : trans isomers [9,10]. The ratio
of the signal intensities at  115–116 ppm and at 107 ppm revealed that the oligomer
fraction contains approximately 20% procyanidin (PC): and 80% prodelphinidin (PD)
[10]. The presence of gallate units was obvious by the carbon chemical shift at  110, 122
and 139 ppm as well as the carbonyl carbon chemical shift at  166 ppm [11].

The mean average molecular size of the oligomers was estimated to be 5–6 flavan-3-ol

units by integration of C-3 signals of the extender units at 73 ppm and the
corresponding signal of the lower flavan-3-ols at 68 ppm [12].

To elucidate the structure in more detail, the oligomer (AWF) was degraded in the

presence of phloroglucinol under acidic conditions at ambient temperature for 30 min
[13,14].

The reaction resulted in the cleavage of the terminal flavonoid units, which were

identified as epigallocatechin, gallocatechin and relatively low amounts of epicatechin,
catechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate. Among the mono-
meric phloroglucinol-captured products were epigallocatechin-(4
! 2)-phloroglucinol
and gallocatechin-(4 ! 2)-phloroglucinol as the main monomeric adducts in
addition

to

epigallocatechin-3-O-gallate-(4
! 2)-phloroglucinol,

epicatechin-3-O-

gallate-(4
! 2)-phloroglucinol and small amounts of epicatechin-(4
! 2)-phloroglu-
cinol; this figure is consistent with the

13

C NMR data. The structures of the flavan-3-ols

and the monomeric phloroglucinol adducts were identified on the basis of 1D- and
2D-NMR (HSQC, HMBC) experiments of their peracetylated derivatives. Comparison
of the data with authentic samples from earlier work and published values identified
these compounds as such [15–21].

To establish the nature of the interflavonoid linkages in the polymer, it was

necessary to isolate some larger scission products which contained these linkages.
Three compounds were isolated by column chromatography alternating between
Sephadex LH-20 and high porosity polystyrene polymer (MCI CHP 20P) using
aqueous methanol solutions. The dimeric phloroglucinol adduct gallocatechin-
(4 ! 8)-epigallocatechin-(4
! 2)-phloroglucinol (compound 1) was isolated as the
main dimer in addition to epigallocatechin-3-O-gallat-(4
! 8)-epigallocatechin-3-O-
gallat-(4
! 2)-phloroglucinol (compound 2) and small amounts of the prodelphinidin
epigallocatechin-(4
! 8)-epigallocatechin.

The structures of compounds 1 and 2 were determined on the basis of their 1D- and

2D-NMR (HSQC, HMBC), CD and ½

20
D

data of their peracetylated derivatives.

The structure of epigallocatechin-(4
! 8)-epigallocatechin was identified by

1

H NMR,

MALDI-TOF-MS and CD spectroscopy of the peracetylated derivative in comparison
with published data [4].

Compound 1 showed a prominent quasi-molecular ion peak at m/z 1384 [M þ Na]

þ

in the MALDI-TOF-MS of its peracetate (compound 1a), which suggests a B-type
diflavonoid constitution composed of two (epi)gallocatechin units and one phloroglu-
cinol moiety. The

1

H NMR of 1a was very similar to that of the analogous dimeric

prodelphinidin gallocatechin-(4 ! 8)-epigallocatechin peracetate [16]. Due to the meta
coupling protons of the additional phloroglucinol ring (G-ring), the peracetate of 1a
showed two additional proton doublets of  6.79 and 6.93 ppm, respectively. The

1

H

NMR of 1a in CDCl

3

(600 MHz) gave two two-proton singlets at  6.82 and 7.07 ppm

typical of pyrogallol-type-B rings of the constituent flavan-3-ol units. The heterocyclic
coupling constants J

2,3(C)

¼

9.0 Hz and J

2,3(F)

< 2 Hz confirmed the relative 2,3-trans

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and 2,3-cis stereochemistry of the flavan-3-ol units corresponding to a gallocatechin and
epigallocatechin moieties, respectively [15]. The correlation between H-8 (A) and
H-4 (C) to C-8a (A) confirmed the ‘‘upper’’ flavan-3-ol unit as gallocatechin. The
location of the interflavonoid linkages was recognized for 1a by long-range correlations
(HMBC) of H-4 (C) with C-8a (D). This key correlation indicates that the flavan-3-ol
units are C-4/C-8 linked [22]. Compound 1 gave phloroglucinol and epigallocatechin-
(4
! 2)-phloroglucinol as the main degradation products after the reaction with 0.1 M
ethanolic HCl under the conditions employed [23]. These degradation products were
identified

by

co-chromatography

in

comparison

with

authentic

compounds.

The high-amplitude negative Cotton effect at low wavelengths (200–230 nm) in the
CD spectrum of 1a confirmed the absolute configuration of the upper unit as 4S [24,25].
In conjunction with the optical rotation ½

20
D

¼ 

33.6

(c 0.15, MeOH), compound 1

was characterized as gallocatechin-(4 ! 8)-epigallocatechin-(4
! 2)-phloroglucinol.

The structure of epigallocatechin-3-O-gallat-(4
! 8)-epigallocatechin-3-O-gallat-

(4
! 2)-phloroglucinol (compound 2) was established with reverse 2D-NMR methods
of its peracetate (compound 2a). The

1

H NMR of 2a was very similar to that of the

analogous dimeric prodelphinidin epigallocatechin-3-O-gallat-(4
! 8)-epigallocate-
chin-3-O-gallat peracetate [26]. The peracetate of 2a showed two additional proton
doublets of  6.89 and 7.04 ppm, respectively, due to the meta coupling protons of the
additional phloroglucinol ring (I-ring). Compound 2 showed a prominent ion peak at
m

/z 1855 in the MALDI-TOF-MS [M þ Na]

þ

of its peracetate (2a), indicative of a

dimeric proanthocyanidin derivative composed of two gallocatechin/epigallocatechin
units, two galloyl moieties and one phloroglucinol ring. The

1

H NMR of 2a in CDCl

3

(600 MHz) gave two sharp two-proton singlets at  7.16 and 7.29 ppm typical of
pyrogallol-type-B rings of the constituent flavan-3-ol units. The location of the
interflavonoid linkage was recognized for 2a by long-range correlation (HMBC) of
H-4 (C) with C-8a (D) [22]. This key correlation indicates that the flavan-3-ol units are
C-4/C-8 linked. The heterocyclic coupling constants (J

2,3

< 2 MHz) confirmed the

relative 2,3-cis configuration of the ‘‘upper’’ and ‘‘lower’’ constituent units [15].
A diagnostic feature in the

1

H NMR spectrum was the presence of two sharp low-field

two-proton singlets ( 7.44 and 7.46 ppm), attributable to the equivalent protons of two
galloyl groups. The structural elucidation was corroborated by acid-catalysed reaction
in the presence of phloroglucinol to give epigallocatechin-3-O-gallat-(4
! 2)-phlor-
oglucinol as a major additional product. This degradation product was identified by
co-chromatography in comparison with the authentic compound. The high-amplitude
positive Cotton effect at 200–240 nm in the CD spectrum of 2a confirmed the absolute
configuration as 4R [24,25]. In conjunction with the optical rotation ½

20
D

¼ þ

144

(c ¼ 0.12, MeOH), compound 2 was characterized as epigallocatechin-3-O-gallat-
(4
! 8)-epigallocatechin-3-O-gallat-(4
! 2)-phloroglucinol.

To the best of our knowledge, 1 and 2 are described here for the first time as well as

the NMR data of their peracetate derivatives.

The isolation of the dimeric phloroglucinol adducts 1 and 2, in addition to a small

amount of the prodelphinidin dimer epigallocatechin-(4
! 8)-epigallocatechin could be
assumed, in the light of the mild conditions employed in their production. These reflect
the relative frequency of the type of the interflavonoid linkages in the original polymer.
Interflavonoid linkages seem to be predominantly 4 ! 8 bonds.

In conclusion, the results demonstrate that the higher oligomeric proanthocyanidins

accounted for 73% of the total antioxidant activity from C. salvifolius herb extract.

Antioxidant oligomeric proanthocyanidins from

C. salvifolius

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Their composition was elucidated by acid-catalysed cleavage in the presence of
phloroglucinol and NMR spectroscopy.

The flavan-3-ol units in the higher oligomeric mixture showed a great similarity in the

chemical structure to the dimeric proanthocyanidins isolated from the air-dried herb
[4] with the predominance of 2,3-cis-configuration, 3

0

,4

0

,5

0

-trihydroxylated B-rings and

the occurrence of galloylated units. The only difference was the absence of
p

-hydroxybenzoyl moiety as acyl residue at C-3 of the flavan-3-ol units in the higher

oligomeric structure and the occurrence of such derivatives in the low-molecular
polyphenols. In contrast to the proanthocyanidin oligomer of Cistus albidus [27],
the oligomer in C. salvifolius is much more heterogeneous, both in terms of the variety
of terminal and extender flavonoid units.

O

RO

OR

OR

OR

OR

OR

O

OR

OR

OR

OR

RO

OR

OR

OR

RO

2:

R= H

2a: R= -C

O

CH

3

CH

3

O

1:

R= H

1a: R= -C

1

′

6

′

1

′

4

6

4

2

I

4

″

6

″

2

″

1a

1a

H

G

4

″

3

″

1

″

5

′

4

′

3

′

2

′

8a

8

8

6

O

O

C

OR

OR

OR

C

OR

OR

OR

8a

4a

4

3

2

F

E

D

C

B

A

6

4

5

′

4

′

3

′

5

′

4

′

3

′

2

6

′

2

′

6

′

2

′

G

4

3

2

F

E

D

4a

8a

8

6

4

3

C

B

A

OR

RO

OR

O

O

OR

OR

OR

RO

OR

O

O

OR

OR

OR

RO

OR

8

2

3. Experimental

3.1. General experimental procedures

1

H NMR spectra were recorded in CDCl

3

on a Varian Gemini 200 (200 MHz), on

Varian Mercury 400 plus or a Bruker AM 600 (600 MHz) relative to CHCl

3

.

13

C NMR

spectra were recorded at 50, 100 or 150 MHz. CD data were obtained in MeOH on a
Jasco J 600. MALDI-TOF mass spectrometer: LAZARUS II (home built), N2-laser
(LSI VSL337ND) 337 nm, 3 ns pulse width, focus diameter 0.1 mm, 16 kV acceleration
voltage, 1 m drift length, data logging with LeCroy9450A, 2.5 ns sampling time and
expected mass accuracy 0.1%, sample preparation: acetylated compounds were
deposited from a solution in CHCl

3

on a thin layer of 2,5-dihydroxybenzoic acid (DHB)

crystals. Analytical TLC was carried out on aluminium sheets (Kieselgel 60 F

254

,

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0.2 mm, Merck) using acetone–toluene–formic acid (60 : 30 : 10 system A). Compounds
were visualized by spraying with vanillin–HCl reagent and 1% ethanolic FeCl

3

solution.

Preparative TLC was performed on silica gel plates (Kieselgel 60 F

254

, 0.5 mm Merck)

using system A. Acetylation was performed in pyridine–acetic acid anhydride (1 : 1.2)
at ambient temperature for 24 h.

3.2. Materials

Phloroglucinol was obtained from Fluka (Seelze, Germany). Potassium nitrosodisul-
phonate (Fremy’s salt) was purchased from Sigma-Aldrich (Taufkirchen, Germany).
Reagents and solvents were purchased from Roth (Karlsruhe, Germany) or Merck
(Darmstadt, Germany).

3.3. Plant material

Cistus salvifolius

L. was collected in El Majdal (Jordan; 06/2002) and identified in

comparison with authentic C. salvifolius obtained from the Botanical Institute,
University Cologne (Germany). A voucher specimen is deposited at the Herbarium
of the Institut fu¨r pharmazeutische Biologie, Mu¨nster, Germany under PBMS90.

3.4. Quantitative analysis of proanthocyanidins

The content of proanthocyanidins in the three fractions (EAF, MWF, AWF) was
determined photometrically after acid depolymerization to the corresponding
anthocyanidins [28]. In all fractions, 1 mg of the dried sample was dissolved in 10 mL
of a solution of concentrated hydrochloric acid in n-butanol (10 : 90, v/v). The closed
vial containing the solution was mixed vigorously and heated for 90 min in a boiling
water bath. After the solution was cooled to room temperature, the absorbance at
550 nm was measured using a Novaspec II spectrophotometer (Pharmacia LKB,
Uppsala, Sweden). The content of proanthocyanidins (mg cyanidin/L) was calculated
by the molar extinction coefficient of cyanidin (" ¼ 17,360 L mol



1

cm



1

).

3.5. Electron spin resonance (ESR) analysis

For measuring the antioxidant activity of the three fractions, 1 mg from each fraction
was dissolved in 1 mL methanol. Aliquots (500 mL) were then allowed to react with an
equal volume of Fremy’s salt (1 mM in phosphate buffer pH 7.4). The ESR spectrum of
Fremy’s radical was obtained after 20 min, by which time the reaction was completed.
Signal intensity was obtained by integration, and the antioxidant activity, expressed as
moles Fremy’s salt reduced by one mole antioxidant, was calculated by comparison
with a control reaction with methanol. Spectra were obtained at 21

C on a Miniscope

MS 100 spectrometer (Magnettech, Berlin, Germany). The microwave power
and modulation amplitude were set at 10 dB and 1500 mG, respectively. For the
measurement, 50 mL of the reaction mixture was added in a micropipette.

Antioxidant oligomeric proanthocyanidins from

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3.6. Extraction and isolation

Air-dried material (2 kg) was exhaustively extracted with acetone–water (7 : 3, 18 L) and
the combined extracts evaporated in vacuo to 1.5 L, filtered to remove the precipitated
chlorophyll, concentrated and defatted with petroleum benzene at 30–50

C. Successive

extractions with ethyl acetate (7.5 L) followed by the evaporation of solvent solid
yielded 25.5 g EAF. The remaining water phase was evaporated to dryness (250 g).
A portion (150 g) of the water phase was successively applied to CC on Sephadex LH-20
(55  900 mm) with 20 L MeOH–H

2

O (1 : 1) until the eluent was colourless

(MWF; 97 g); then acetone–water (3 : 7) was used to elute the oligomeric fraction
(AWF; 53 g).

3.7. Degradation with phloroglucinol

The oligomeric fraction (AWF) of C. salvifolius obtained as described above (15 g)
was treated for 30 min at room temperature with phloroglucinol (10 g) in 1% HCl in
EtOH (50 mL) under continuous shaking [13,14]. The solution was concentrated at
reduced pressure (24.9 g). A portion (20 g) was fractionated on Sephadex LH-20
(55  900 mm) using EtOH (3 L), EtOH–MeOH 1 : 1 (6.5 L) and acetone–water 7 : 3
(2.5 L) to give 12 fractions (frs.). Fraction 2 (1140–3720 mL, 117 mg) was subjected to
chromatography on MCI-gel CHP 20P (25  250 mm) with a 10–80% MeOH linear
gradient (17 mL/fr.) to afford catechin (subfrs. 20–44, 39 mg) and epicatechin
(subfrs. 48–59, 60 mg).

Fraction 3 (3720–4900 mL, 683 mg) was separated on MCI-gel with the same gradient

as above to afford (subfrs.12–19, 126 mg) gallocatechin and (subfrs. 27–45, 361 mg)
epigallocatechin. Fraction 4 (4900–5200 mL, 56 mg) was separated on MCI-gel
to afford (subfrs. 23–31, 19 mg) epicatechin-(4
! 2)-phloroglucinol. Fraction 5
(5200–5600 mL, 1.6 g) was separated on MCI to afford epigallocatechin-(4
! 2)-
phloroglucinol (33–50, 807 mg). Gallocatechin-(4 ! 2)-phloroglucinol was isolated
from fr. 6 (5600–6200 mL, 572 mg) and MCI-gel chromatography (subfrs. 42–60,
393 mg). Fraction 7 (6200–6700 mL, 250 mg) was subjected to chromatography
on

MCI-gel

elution

to

afford

epicatechin-3-O-gallat-(4
! 2)-phloroglucinol

(subfrs.

71–79,

127 mg)

and

epicatechin-3-O-gallat

(subfrs.

37–42,

22 mg).

Fraction 8 (6700–7200 mL, 262 mg) was separated as above to yield epigallocatechin-
3-O-gallat-(4
! 2)-phloroglucinol (subfrs. 59–67, 79 mg), epigallocatechin-3-O-gallat
(subfrs.

19–22,

37 mg)

and

epigallocatechin-(4
! 8)-epigallocatechin

(subfrs.

47–55, 11 mg).

All compounds were identified after acetylation by comparing their physical data

(NMR, MS, and CD) with those of authentic samples and published values [4,15–21].

3.8. Gallocatechin-(4a ! 8)-epigallocatechin-(4b ! 2)-phloroglucinol (1)

Fraction 9 (7200–7750 mL, 132 mg) obtained from Sephadex LH-20 column was
subjected to chromatography on MCI-gel CHP 20P (25  450 mm) with a 20–60%
MeOH

linear

gradient

(17 mL/subfr.)

to

afford

an

amorphous

powder

(subfrs. 34–45, 99 mg) 1: ½

20
D

¼ 

33.6

(c 0.15, MeOH). Fifty milligrams were

acetylated to give 1a: MALDI-TOF-MS: [M þ Na]

þ

m/z

1384.

1

H NMR (CDCl

3

,

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600 MHz):  1.42–2.44 (m, OAc),  4.24 [d, J ¼ 3.8 Hz, H-4 (F)],  4.83 [d, J ¼ 10.0 Hz, H-4
(C)],  4.90 [d, J ¼ 9.0 Hz, H-2 (C)],  5.23 [dd, J ¼ 3.8 and 1.1 Hz, H-3 (F)],  5.68 [dd,
J ¼

9.0 and 10.0 Hz, H-3 (C)],  6.39 [d, J ¼ 2.3 Hz, H-6 (A)],  6.55 [d, J ¼ 2.3 Hz, H-8

(A)],  6.65 [s, H-6 (D)],  6.79 [d, J ¼ 2.3 Hz, H-4 or H-6 (G)],  6.82 [s, H-2

0

/H-6

0

(E)],



6.93 [d, J ¼ 2.3 Hz, H-6 or H-4 (G)],  7.07 [s, H-2

0

H-6

0

(B)].

13

C NMR (CDCl

3

,

150 MHz):  30.9 [C-4 (F)],  34.6 [C-4 (C)],  70.2 [C-3 (F)],  72.1 [C-3 (C)],  74.2
[C-2 (F)],  80.0 [C-2 (C)], 108.1 [C-8 (A)],  109.8 [C-6 (D)],  110.2 [C-6 (A)], 113.0 [C-4a
(D)],  114.2 [C-4 or C-6 (G)],  115.2 [C-6 or C-4 (G)],115.6 [C-4a (A)],  117.5 [C-8 (D)],


118.9 [C-2

0

/C-6

0

(E)],  120.0 [C-2

0

/6

0

(B)],  120.1 [C-2 (G)],  134.2 [C-1

0

(E)],  134.4

[C-1

0

(B)],  134,8 [C-4

0

(E)],  135.0 [C-4

0

(B)],  143.2 [C-3

0

, C-5

0

(B) and C-3

0

, C-5

0

(E)],



148.5–149.5 [C-5 (A),C-7 (A), C-5 (D), C-7 (D), C-1 (G), C-3 (G) and C-5 (G)],



153.0 [C-8a (D)],  156.3 [C-8a (A)]. After the reaction of compound 1 (20 mg) in 0.1 N

ethanolic HCl (1 mL) [23], the mixture solution was concentrated under a stream of N

2

to

dryness and purified on prep. TLC in system A. The two main degradation products
were further purified on preparative TLC on cellulose (t-BuOH–CH

3

COOH–H

2

O;

60 : 20.20) to yield epigallocatechin-(4
! 2)-phloroglucinol (6.5 mg) and phloroglucinol
(6.1 mg).

3.9. Epigallocatechin-3-O-gallat-(4b ! 8)-epigallocatechin-3-O-gallat-(4b ! 2)-

phloroglucinol (2)

Fraction 10 (7750–8100 ml, 81 mg) achieved from Sephadex LH-20 column was
subjected to chromatography on MCI-gel CHP 20P (25  450 mm) with a 20–60%
MeOH linear gradient (17 mL/subfr.) to afford an amorphous powder (subfrs. 24–37,
31 mg). 2:½

20
D

¼ þ

144

(c ¼ 0.12, MeOH). Twenty milligrams were acetylated to give 2a:

MALDI-TOF-MS: [M þ Na]

þ

m/z

1855.

1

H NMR (CDCl

3

, 600 MHz):  1.23–2.30

(m, OAc),  4.61 [d, J ¼ 1.8 Hz, H-4 (F)],  4.93 [brs, H-4 (C)],  5.37 [brs, H-2 (C)],  5.56
[m, H-3 (F)],  5.73 [d, m, H-3 (C)],  5.74 [brs, H-2 (F)],  6.64 [s, H-6 (D)],


6.71 [d, J ¼ 2.3 Hz, H-6 (A)],  6.77 [d, J ¼ 2.3 Hz, H-8 (A)],  6.89 [d, J ¼ 2.3 Hz, H-4

or H-6 (I)],  7.04 [d, J ¼ 2.3 Hz, H-6 or H-4 (I)],  7.16 [s, H-2

0

/H-6

0

(E)],  7.29 [s, H-2

0

/

H-6

0

(B)],  7.44 [s, H-2

00

/H-6

00

(G)],  7.46 [s, H-2

00

/H-6

00

(H)].

13

C NMR (CDCl

3

,

150 MHz):  34.9 [C-4 (C)],  35.0 [C-4 (F)],  71.0 [C-3 (F)],  72.4 [C-3 (C)],  74.3 [C-2
(F)],  74.6 [C-2 (C)],  107.7 [C-8 (A)],  109.5 [C-6 (A)],  110.8 [C-4a (A)],  111.3 [C-6
(D) and C-4a (D) ,  114.5 [C-4 or C-6 (I)],  115.1 [C-6 or C-4 (J)],  116.3 [C-8 (D)],


117.9 [C-2

0

/C-6

0

(E)],  119.3 [C-2

0

/C-6

0

(B)],  119.9 [C-2 (I))],  122.3 [C-2

00

/C-6

00

(H)],



122.5 [C-2

00

/6

00

(G)],  127.1 [C-1

00

(G) and C-1

00

(H)],  134.2–134.6 [C-1

0

(B), C-1

0

(E),

C-4

0

(B) and C-4

0

(E],  139,0 [C-4

00

(G) or C-4

00

(H)],  139.1 [C-4

00

(H) or C-4

00

(G)],



143.2–143.4 [C-3

0

(B), C-5

0

(B), C-3

0

(E), C-5

00

(E), C-3

00

(G), C-5

00

(G), C-3

00

(H) and

C-5

00

(H)],  148.6–150.1 [C-5 (A),C-7 (A), C-5 (D), C-7 (D), C-1 (I), C-3 (I) and C-5 (I)],



152.2 [C-8a (D)],  154.9 [C-8a (A)],  162.1 [C-1a (G)],  163.4 [C-1a (H)].

Purified proanthocyanidin (10 mg, compound 2) was made to reacted with

phloroglucinol (10 mg) in 1% HCl in EtOH (1 mL) for 15 min at room temperature.
with continuous shaking. The solution was then concentrated under a stream of N

2

to

dryness and purified on preparative TLC in system A. The main phloroglucinol adduct
was further purified on prep. TLC on Cellulose (t-BuOH–CH

3

COOH–H

2

O; 60 : 20.20)

to yield epigallocatechin-3-O-gallat-(4 ! 2)-phloroglucinol (4.7 mg).

Antioxidant oligomeric proanthocyanidins from

C. salvifolius

1223

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Acknowledgments

F. Qa’dan would like to acknowledge the DAAD and the Deanship of Research at the
University of Petra for funds and grants (Grant No.1/5/2002). We wish to acknowledge
also the help of Dr H. Lahl, Ms. M. Heim (Inst. f. Pharmazeutische Chemie, Mu¨nster)
and Dr. Brian Lockwood (School of Pharmacy, Manchester) for the NMR spectra,
Dr H. Luftmann (Inst. f. Organische Chemie, Muenster) for the MALDI-MS spectra,
Prof. Dr V. Buß (Theoretische Chemie, Duisburg) for the CD spectra and Ms Meike
Bergmann (BMP Laboratory, Berlin) for the ESR spectra.

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