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
yFaculty of Pharmacy, The University of Petra, PO Box 961343, Amman, Jordan
zInstitut fuer Pharmazeutische Biologie und Phytochemie, Hittorfstrasse 56,
48149 Muenster, Germany
xFaculty 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
13
resulting from the degradation with phloroglucinol were confirmed by 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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Antioxidant oligomeric proanthocyanidins from C. salvifolius 1217
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.
Proanthocyanidin Antioxidant activity
concentration (mmol Fremy s salt Percentage of
Fraction (mg cyanidin L 1 fraction) reduced L 1 fraction) antioxidant activity (%)
Ethyl acetate (EAF) 465 16a 9.7 0.1 9.0
Methanol H2O (MWF) 562 21 19.2 0.4 18.0
Acetone H2O (AWF) 3720 63 78 1.5 73.0
a
Data are mean SD obtained from three different assays.
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1218 F. Qa dan et al.
13
close to 77 ppm and 84 ppm by C NMR spectroscopy (solvent: MeOH-d4, 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-
13
cinol; this figure is consistent with the 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 Š20 data of their peracetylated derivatives.
D
1
The structure of epigallocatechin-(4 ! 8)-epigallocatechin was identified by 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. The1H 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. The1H
NMR of 1a in CDCl3 (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 J2,3(C) ź 9.0 Hz and J2,3(F) < 2 Hz confirmed the relative 2,3-trans
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Antioxidant oligomeric proanthocyanidins from C. salvifolius 1219
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 Š20 ź 33.6 (c 0.15, MeOH), compound 1
D
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
1
of its peracetate (compound 2a). The 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
1
units, two galloyl moieties and one phloroglucinol ring. The H NMR of 2a in CDCl3
(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 (J2,3 < 2 MHz) confirmed the
relative 2,3-cis configuration of the   upper  and   lower  constituent units [15].
1
A diagnostic feature in the 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 Š20 źþ144
D
(c ź 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.
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1220 F. Qa dan et al.
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, 30,40,50-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.
OR
OR
32
OR 32
22
OR
22
42
8 B
42
8a 12 8 B
RO O
8a 12
RO O
2 OR
52 OR
52 OR
C 2
A 62
3 33
62
C
A
4 3
6 1a
4a 4
6
OR 13
43
4a
O C G OR
OR
OR
OR
O
OR
OR
32
OR
42
22 OR
E
8
E
8
RO O
RO 8a O
52 OR
2
OR OR
D F 62
3 D F
23
4
1a
4
OR 43
O H
C OR
OR
OR
63
O
OR
2
2
RO OR
RO OR
G
I
6
4
6 4
OR
OR
1: R= H
2: R= H
1a: R= -C CH3
2a: R= -C CH3
O
O
3. Experimental
3.1. General experimental procedures
1
H NMR spectra were recorded in CDCl3 on a Varian Gemini 200 (200 MHz), on
13
Varian Mercury 400 plus or a Bruker AM 600 (600 MHz) relative to CHCl3. 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 CHCl3 on a thin layer of 2,5-dihydroxybenzoic acid (DHB)
crystals. Analytical TLC was carried out on aluminium sheets (Kieselgel 60 F254,
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Antioxidant oligomeric proanthocyanidins from C. salvifolius 1221
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 FeCl3 solution.
Preparative TLC was performed on silica gel plates (Kieselgel 60 F254, 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 fur pharmazeutische Biologie, Munster, 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 (" ź 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.
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1222 F. Qa dan et al.
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 H2O (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 Š20 ź 33.6 (c 0.15, MeOH). Fifty milligrams were
D
1
acetylated to give 1a: MALDI-TOF-MS: [M þ Na]þ m/z 1384. H NMR (CDCl3,
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Antioxidant oligomeric proanthocyanidins from C. salvifolius 1223
600 MHz): 1.42 2.44 (m, OAc), 4.24 [d, J ź 3.8 Hz, H-4 (F)], 4.83 [d, J ź 10.0 Hz, H-4
(C)], 4.90 [d, J ź 9.0 Hz, H-2 (C)], 5.23 [dd, J ź 3.8 and 1.1 Hz, H-3 (F)], 5.68 [dd,
J ź 9.0 and 10.0 Hz, H-3 (C)], 6.39 [d, J ź 2.3 Hz, H-6 (A)], 6.55 [d, J ź 2.3 Hz, H-8
(A)], 6.65 [s, H-6 (D)], 6.79 [d, J ź 2.3 Hz, H-4 or H-6 (G)], 6.82 [s, H-20/H-60 (E)],
13
6.93 [d, J ź 2.3 Hz, H-6 or H-4 (G)], 7.07 [s, H-20H-60 (B)]. C NMR (CDCl3,
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-20/C-60 (E)], 120.0 [C-20/60 (B)], 120.1 [C-2 (G)], 134.2 [C-10 (E)], 134.4
[C-10 (B)], 134,8 [C-40 (E)], 135.0 [C-40 (B)], 143.2 [C-30, C-50 (B) and C-30, C-50 (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 N2 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 CH3COOH H2O;
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 Š20 źþ144 (c ź 0.12, MeOH). Twenty milligrams were acetylated to give 2a:
D
1
MALDI-TOF-MS: [M þ Na]þ m/z 1855. H NMR (CDCl3, 600 MHz): 1.23 2.30
(m, OAc), 4.61 [d, J ź 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 ź 2.3 Hz, H-6 (A)], 6.77 [d, J ź 2.3 Hz, H-8 (A)], 6.89 [d, J ź 2.3 Hz, H-4
or H-6 (I)], 7.04 [d, J ź 2.3 Hz, H-6 or H-4 (I)], 7.16 [s, H-20/H-60 (E)], 7.29 [s, H-20/
13
H-60 (B)], 7.44 [s, H-200/H-600 (G)], 7.46 [s, H-200/H-600 (H)]. C NMR (CDCl3,
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-20/C-60 (E)], 119.3 [C-20/C-60 (B)], 119.9 [C-2 (I))], 122.3 [C-200/C-600 (H)],
122.5 [C-200/600 (G)], 127.1 [C-100 (G) and C-100 (H)], 134.2 134.6 [C-10 (B), C-10 (E),
C-40 (B) and C-40 (E], 139,0 [C-400 (G) or C-400 (H)], 139.1 [C-400 (H) or C-400 (G)],
143.2 143.4 [C-30 (B), C-50 (B), C-30 (E), C-500 (E), C-300 (G), C-500 (G), C-300 (H) and
C-500 (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 N2 to
dryness and purified on preparative TLC in system A. The main phloroglucinol adduct
was further purified on prep. TLC on Cellulose (t-BuOH CH3COOH H2O; 60 : 20.20)
to yield epigallocatechin-3-O-gallat-(4 ! 2)-phloroglucinol (4.7 mg).
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1224 F. Qa dan et al.
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, Munster)
¨
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.
References
[1] R.A. Farley, T. McNeilly. Hereditas, 132, 183 (2000).
[2] S. Al-Khalil. Int. J. Pharmacogn., 33, 317 (1995).
[3] E. Yesilada, I. Gurbuz, H. Shibata. J. Ethnopharmacol., 66, 289 (1999).
[4] A. Danne, F. Petereit, A. Nahrstedt. Phytochemistry, 37, 533 (1994).
[5] F. Petereit. Polyphenolische Inhaltsstoffe und Untersuchungen zur entzuendungshemmenden
Aktivitaeten der traditionellen Arzneipflanze Cistus incanus L. (Cistaceae). PhD thesis, University
Muenster (Germany) (1992).
[6] A. Dauer, H. Rimpler, A. Hensel. Planta Medica, 69, 89 (2003).
[7] A. Danne. Polyphenole aus den traditionellen Arzneipflanzen Cistus incanus L. und Cistus salvifolius
L. PhD thesis, University Muenster (Germany) (1994).
[8] L.J. Porter. Tannins. In Methods in Plant Biochemistry, J.B. Harborne (Ed.), p. 389, Academic Press,
San Diego (1989).
[9] T. Eberhardt, R.A. Young. J. Agric. Food Chem., 42, 1704 (1994).
[10] R.H. Newman, L.J. Porter, L.Y. Foo, S.R. Johns, R.I. Willing. Magn. Reson. Chem., 25, 118 (1987).
[11] D. Sun, H. Wong, L.Y. Foo. Phytochemistry, 26, 1825 (1987).
[12] Z. Czochanska, L.Y. Foo, R.H. Newman, L.J. Porter. J. Chem. Soc. Perkin Trans. (I), 2278 (1980).
[13] L.Y. Foo, J.J. Karchesy. Phytochemistry, 28, 3185 (1989).
[14] L.Y. Foo, R. Newman, G. Waghorn, W.C. McNabb, M.J. Ulyatt. Phytochemistry, 41, 617 (1996).
[15] A.C. Fletcher, L.J. Porter, E. Haslam, R.K. Gupta. J. Chem. Soc. Perkin Trans. (I), 14, 1628 (1977).
[16] L.Y. Foo, L.J. Porter. J. Chem. Soc. Perkin Trans. (I), 10, 1186 (1978).
[17] L.J. Porter, R.H. Newman, L.Y. Foo, H. Wong, R.W. Hemingway. J. Chem. Soc. Perkin Trans. (I),
1217 (1982).
[18] L.Y. Foo, L.J. Porter. J. Chem. Soc. Perkin Trans. (I), 1535 (1983).
[19] F. Qa dan. Analytik oligomerer proanthocyanidine aus Cistus albidus (Cistaceae). PhD thesis,
University Muenster (Germany) (1999).
[20] J.A. Kennedy, P.J. Graham. J. Agric. Food Chem., 49, 1740 (2001).
[21] O. Ploss, F. Petereit, A. Nahrstedt. Pharmazie, 56, 506 (2001).
[22] L. Balas, J. Vercauteren. Magn. Reson. Chem., 32, 386 (1994).
[23] R.S. Thompson, D. Jacques, E. Haslam, J.N. Tanner. J. Chem. Soc. Perkin Trans. (I), 1387 (1972).
[24] M.W. Barrett, W. Klyne, P.M. Scopes, A.C. Fletcher, L.J. Porter, E. Haslam. J. Chem. Soc. Perkin.
Trans. (I), 2375 (1979).
[25] J.J. Botha, D.A. Young, D. Ferreira, D.G. Roux. J. Chem. Soc. Perkin Trans. (I), 1213 (1981).
[26] J.P. De Mello, F. Petereit, A. Nahrstedt. Phytochemistry, 41, 807 (1996).
[27] F. Qa dan, F. Petereit, A. Nahrstedt. Pharmazie, 58, 416 (2003).
[28] M.S. Nikfardjam. Polyphenole in Weissweinen und Traubensaeften und ihre Veraenderungen im Verlauf
der Herstellung. PhD thesis, University Giessen (Germany) (2001).
Downloaded by [Politechnika Poznanska] at 02:14 20 June 2016