Ericifolin: An eugenol 5-O-galloylglucoside and other phenolics from Melaleuca ericifolia

Article abstract of DOI:10.1016/j.phytochem.2007.03.010

Ericifolin, an eugenol 5-O-β-(6′-O-galloylglucopyranoside) possessing the naturally unknown phenolic moiety, 5-hydroxyeugenol, together with the two new phenolics, 2-O-p-hydroxybenzoyl-6-O-galloyl-(α/β)-⁴C₁-glucopyranose and 3-methoxyellagic acid 4-O-rhamnopyranoside have been isolated from the antibacterial leaves extract of Melaleuca ericifolia. In addition, 19 known phenolics were also separated and characterized. All structures were elucidated on the basis of analysis of ¹H, ¹³C NMR, HMQC, HMBC and FTMS spectral data.

Full text of DOI:10.1016/j.phytochem.2007.03.010

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 1464–1470
www.elsevier.com/locate/phytochem
Ericifolin: An eugenol 5-O-galloylglucoside and other phenolics
from Melaleuca ericifolia
a
a
a
b
c
S.A.M. Hussein , A.N.M. Hashim , R.T. El-Sharawy , M.A. Seliem , M. Linscheid ,
d
a,
*
U. Lindequist , M.A.M. Nawwar
a
Department of Phytochemistry and Chemosystematic, National Research Center, Dokki, Cairo, Egypt
b
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Egypt
Department of Chemistry, Humboldt-Universit a¨ t zu Berlin, Brook Taylor Strasse 2, 12489 Berlin, Germany
Institut f u¨ r Pharmazie, Pharmazeutische Biologie, Ernst-Moritz-Arndt-Universit a¨ t, Greifswald, D-17487 Greifswald, Germany
c
d
Received 8 September 2006; received in revised form 6 March 2007
Available online 20 April 2007
Abstract
0
Ericifolin, an eugenol 5-O-b-(6 -O-galloylglucopyranoside) possessing the naturally unknown phenolic moiety, 5-hydroxyeugenol,
4
together with the two new phenolics, 2-O-p-hydroxybenzoyl-6-O-galloyl-(a/b)- C -glucopyranose and 3-methoxyellagic acid 4-O-
1
rhamnopyranoside have been isolated from the antibacterial leaves extract of Melaleuca ericifolia. In addition, 19 known phenolics were
1
13
also separated and characterized. All structures were elucidated on the basis of analysis of H, C NMR, HMQC, HMBC and FTMS
spectral data.
ꢀ
2007 Elsevier Ltd. All rights reserved.
Keywords: Myrtaceae; Antimicrobial; Melaleuca ericifolia; Phenolics; Ericifolin; 2-O-p-hydroxybenzoyl-6-O-galloyl glucose; 3-Methoxyellagic acid
-rhamnoside
4
1
. Introduction
(Seligmann and Wagner, 1981; Bouchet et al., 1998). Mel-
aleuca ericifolia Sm. in particular produces essential oil
which possesses antimicrobial, antifungal and antiviral
activities. The oil also exhibited a marked antioxidant
effect that improve the parameters of vitamins E and C
as well as superoxide dismutase enzyme, thus can be used
as a free radical scavenger (Farag et al., 2004). As long as
the available literature is concerned this plant has not
been subjected to any previous phytochemical investiga-
tion of its constitutive phenolics. Due to our interest in
biological activity as well as the diverse phenolic metabo-
lites production of the Myrtaceous plants, investigations
of the activity against multiresistant gram-positive and
gram-negative bacteria and of the chemistry of the pheno-
lic constituents in M. ericifolia were undertaken during
the course of the present work. The plant is a large shrub
or a small tree of 20 feet height. Its leaves are narrowly
linear or nearly cylindrical of 1/2 in. or less long, while
the flowers are yellowish white in cylindrical spikes of
Despite the valuable discoveries made through studies
of constitutive phenolics of several Myrtaceous species
Moharram et al., 2003; El-Toumy et al., 2001; Seligmann
(
and Wagner, 1981), many of these plants remain virtually
unexplored from a chemical stand point of view. Thus, we
have initiated studies of two of these plants, namely,
Eugenia edulis Vell. and Eugenia jambos L. as potential
sources of new bioactive phenolics (Hussein et al., 2002;
Hussein et al., 2003). Among the Myrtaceous genera,
the genus Melaleuca, also known as bottle-brush com-
prises over 250 species of Australian trees and shrubs
(
Baily, 1958). They provided extracts which possess anti-
septic, analgesic, vermifuge and antioxidant activities
*
Corresponding author. Tel.: +20 2 2752711; fax: +20 2 3370931.
E-mail addresses: mahmoudnawwarhesham@yahoo.com, Mahmoud
AMNawwar@netscape.net (M.A.M. Nawwar).
0
031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.03.010

S.A.M. Hussein et al. / Phytochemistry 68 (2007) 1464–1470
1465
1
/2 to 1 in. long. In the present study, chemical studies of
bated with b-glucosidase enzyme (lyophilized chromato-
graphically pure, salts free enzyme from Almond,
BDH) for 24 h, at 37 ꢁC, but it was partially hydrolysed
(0.1 N aqueous HCl, 1/4 h, 100 ꢁC) to 6-O-galloyl glu-
cose (Nawwar and Hussein, 1994a) and 6a). The latter
presented UV maxima which were reminiscent of a phe-
the aqueous alcohol extract of leaves of this plant led to
the isolation and structure determination of 22 phenolic
compounds including three new phenolics, namely, euge-
0
nol 5-O-b-(6 -O-galloylglucopyranoside) which we named
ericifolin (6), 2-O-p-hydroxybenzoyl-6-O-galloyl-(a/b)-
4
+
C -glucopyranose (11) and 3-methoxyellagic acid 4-O-a-
nyl propene system and exhibited an [M] molecular ion
1
1
L-rhamnopyranoside (17).
at m/z 180 in its EI-MS spectrum. The H NMR spec-
trum of 6a was in consistence with a 3,4,5-trisubstituted
phenyl propene structure (Table 2). These data suggested
1
3
2
. Results and discussion
that 6a is the 5-hydroxy derivative of eugenol. In the
C
NMR spectrum of 6a, the propenyl side chain was recog-
nized from the resonances at d 39.9 (C-7), 137.5 (C-8)
and 115.4 (C-9). The aromatic resonances were assigned
(Table 2) through the substituent additive rules applied
when introducing a hydroxyl substituent at C-5 of the
eugenol molecule. Final confirmation of the identity of
6a as 5-hydroxy eugenol was achieved through synthesis
(Rao et al., 1949). The parent compound, ericifolin (6)
had the molecular formula C H O as indicated by
During evaluation of its biological activity (Cooksey
et al., 1993; Sleigh and Timburg, 1981), M. ericifolia
showed a capacity [minimal inhibitory concentrations
MIC), see Section 3] to inhibit the growth of several
gram-positive and gram-negative bacteria, among them
Staphylococcus aureus. The test bacterial strains were
selected because of their resistance properties and of their
medical relevance. Besides, the activity of the pure new
compounds 6, 11 and 17 have also been evaluated (Table
(
2
3
26 12
ꢀ
ESIFTMS ([MꢀH] : 493.1361, calc.: 493.1351). In the
1
1
), whereby compound 17 has shown moderate inhibiting
H NMR spectrum of 6 the resonance of the aromatic
effects against the multiresistant bacterial strain S. aureus
Norddeutscher Epidemiestamm. The remarkable antimi-
crobial effect of the extract could be due to the existing
combination of constituents or it could be attributed to
other compound(s) than 6, 11 and 17.
Following column chromatographic fractionation of the
antibacterial extract obtained by extraction of the leaves of
M. ericifolia by aqueous alcohol, 22 compounds (1–22)
were isolated. Conventional and spectral analysis mainly
by NMR spectroscopy and by mass spectrometry indicated
that three of these compounds are new natural products (6,
proton H-6 at d ppm 6.43, when compared with the cor-
responding resonance in the spectrum of free 5-hydroxy-
eugenol (Table 2) was found to be shifted downfield
(Dd = 0.13 ppm), thus confirming O-glucosidation, at
OH-5. The spectrum also showed a distinct anomeric
glucose proton resonance at d 4.7 and a pair of glucose
proton resonances at d 4.45 and 4.39 assignable to the
two methylenic glucose protons whose geminal hydroxyl
group is galloylated. Direct correlations observable in the
HMQC and HMBC spectra allowed unambiguous
assignment of the protonated carbons and enabled deter-
mination of site of attachments of the different moieties
1
1 and 17).
3
Ericifolin (6), an off-white amorphous powder, showed
in the molecule of 6 (see Table 2). Among the J corre-
chromatographic properties and color reactions (positive
FeCl3 and KIO3 tests) specific for galloyl esters (Had-
dock et al., 1982). Normal acid hydrolysis of 6 (2 N
aqueous HCl, 2 h, 100 ꢁC) yielded glucose, gallic acid
and a third compound 6a, which was separated by pre-
parative paper chromatography (Prep. PC). The parent
compound 6 was recovered unchanged after being incu-
lations recognized in the HMBC spectrum of 6, one was
found correlating the methoxyl proton signal at d 3.69 to
the aromatic carbon signal of C-3 at d 148.0, another
correlated the anomeric glucose proton at d 4.7 to the
aromatic carbon signal of C-5 at d 145.6 and a third cor-
related the methylenic glucose protons at d 4.45 to the
00
carbonyl carbon C-7 at d 165.9. These and the above
Table 1
Activity of the aqueous alcohol leaf extract and compounds 6, 11 and 17) of Melaleuca ericifolia against multiresistant bacteria
Bacterial strains
Test sample
Inhibition zones (mm, including paper disc) against
Staphylococcus aureus Norddeutscher
S. aureus
34289
S. epidermidis
847
S. h a¨ molyticus
535
Pseudomonas
aeruginosa 595
Epidemiestamm
Melaleuca ericifolia
20
14
16
8
10
(2 mg/disc)
Compound 6 (2 mg/disc)
Compound 11 (2 mg/
disc)
8
8
r
r
r
r
r
r
r
r
Compound 17 (2 mg/
disc)
14
10
12
r
8
Control (30 lg/disc)
Amikacin 22
Amikacin 22
Amikacin 20
Amikacin 18
Doxycyclin 16
r, no activity.

1466
S.A.M. Hussein et al. / Phytochemistry 68 (2007) 1464–1470
Table 2
NMR spectral data of ericifolin (6), 5-hydroxyeugenol (6a), 3-methoxyellagic acid 4-O-a-L-rhamnopyranoside (17) and 3-methoxyellagic acid (17a)
6
6a
a
d
H
(J, Hz)
d
C
HMBC
d
H
(J, Hz)
d
C
1
2
3
4
5
6
7
8
9
9
1
2
3
4
5
6
6
1
2
3
4
5
6
7
130.0 s
109.3 d
148.0 s
134.34 s
145.6 s
107.3 d
39.2 t
130.7
103.5
147.3
131.4
143.8
108.9
39.9
6.45 (br s, Dm1/2 = 4)
4, 6, 7, 2, 3, 5weak
6.46 (br s,m Dm1/2 = 4)
6.43 (br s, Dm1/2 = 4)
3.02 (8)
5.72 (m)
4.9 (14)
4.8 (br s, Dm1/2 = 4)
4.7 (7.5)
3.30–3.70 (m)
3.30–3.70 (m)
3.30–3.70 (m)
3.30–3.70 (m)
4.45 (12)
2, 4, 6, 7, 1weak, 5weak
6.30(br s, Dm1/2 = 4)
3.26(8)
5.92(m)
5.09(15)
4.96 (br s, Dm1/2 = 4)
2, 6, 9, 1weak
,
137.9 d
115.4 t
137.5
115.4
a
7
5
b
0
102.5 d
74.11 d
75.6 d
70.00 d
73.4 d
0
0
0
0
0
00
63.58 dd
7
a
0
4.39 (12 and 4.5)
b
0
0
0
0
0
0
0
0
0
0
0
0
0
0
119.3 s
108.7 d
145.8 s
138.8 s
145.8 s
108.7 d
165.9 s
55.87 q
00
00
00
00
6.96 s
4 , 6 , 2 , 7
00
00
00
00
6.96 s
2 , 4 , 6 , 7
OMe
3.69
3
3.81
55.3
1
7
17a
1
a
d
H
(J, Hz)
d
C
and J (C Æ H)
HMBC
d
H
(J, Hz)
d
C
1
2
3
4
5
6
7
1
2
3
4
114.1 s
139.98 s
146.36 s
141.6 s
111.47 d (168 Hz)
111.13 s
158.54 s
112.8 s
135.94 s
141.3 s
152.5 s
111.35 d (165 Hz)
106.8 s
158.50 s
60.84 q (146 Hz)
100.02 d (173 Hz)
69.83 d (142 Hz)
70.00 d (145 Hz)
71.7 d (146 Hz)
69.76 d (141 Hz)
17.75 q (128 Hz)
112.36
141.98
140.64
148.63
111.84
112.66
159.40
112.98
136.63
140.21
152.63
110.83
107.85
159.30
61.45
7.69 s
1, 3, 7, 4(weak)
7.47 s
0
0
0
0
0
0
0
0
0
0
0
0
5
6
7
7.47 s
1 , 3 , 7 , 4
, 6
7.42 s
ðweakÞ
ðweakÞ
OMe
4.03
3
4
4.00
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
5.46 (br s, Dm1/2 = 4)
Me
1.25 (d, J = 6 Hz)
a
H–C.
given data finally confirmed the structure of compound 6
to be eugenol 5-O-b-(6 -galloylglucopyranoside) or ericif-
olin, a new phenolic, which has not been previously
reported to occur in nature.
molecular formula of C H O . On normal acid hydroly-
20 20 12
0
sis 11 yielded glucose, gallic and p-hydroxybenzoic acid
(Hussein et al., 2006), but it was partially hydrolysed to
6-O-galloyl glucose (Nawwar and Hussein, 1994a) and p-
hydroxybenzoic acid, thus suggesting that 11 is a mono-
Compound 11 was obtained as an off-white amorphous
powder and showed color reactions indicative of galloyl
1
O-p-hydroxybenzoyl-6-O-galloyl glucose. The H spectrum
ꢀ
esters (Haddock et al., 1982). It exhibited an [MꢀH]
of 11 (DMSO-d , room temp.) revealed two distinct pat-
6
molecular ion in its ESIFTM mass at m/z = 451.0877
terns of proton signals belonging to di-O-substituted
a- and b-glucose anomers. In this spectrum, a pair of
(
calc.: 451.0871) corresponding to a M of 452 and to a
r

S.A.M. Hussein et al. / Phytochemistry 68 (2007) 1464–1470
1467
1
3
0
doublets, centered at d 5.0 (J = 3.5 Hz) and at d 4.5
J = 8 Hz) were assigned to the a- and b-glucose anomeric
by comparison with the C data reported for 3,3 -dimeth-
oxyellagic acid (Sato, 1987) and for ellagic acid (Nawwar
et al., 1994b) as well. These data confirmed the structure
of 17a to be 3-methoxyellagic acid. Consequently, the par-
ent compound 17 is 3-methoxyellagic acid mono-a-O-L-
rhamnopyranoside.
(
protons, respectively. The pair of downfield glucose proton
signals at d 4.4 and 4.52 were attributed to the glucose H-2
proton in the a- and b-glucose anomers, respectively. Con-
sequently, the p-hydroxybenzyol moiety is present in the
molecule of 11 attached to the glucose carbon C-2. In
Comparison of the 1D NMR data proved that 17 con-
tained a rhamnoside moiety which revealed its anomeric
proton as a broad singlet at d 5.46, of half-width (Dt1/2
= 4 Hz). This finding, together with the result of hydroly-
sis with hesperidinase enzyme proved the a-configuration
addition, the values of the above given coupling constants
4
indicate that the glucose core in 11 is adopting a C -con-
1
formation. The weight of evidences given above, confirmed
that compound 11 is 2-O-p-hydroxybenzoyl-6-O-galloyl-
4
13
1
13
(
a/b)- C -glucopyranose. The C NMR spectrum of 11
of the existing rhamnose moiety. Besides, in a H– C
NMR spectrum of 17, a high J(C-1.H) of 173 Hz was
1
1
contained essentially double signals for most of the glu-
cose, galloyl and p-hydroxybenzoyl carbons. Signals were
assigned by comparison with the C NMR data reported
for 2,6-di-O-galloyl glucose (Nawwar and Hussein,
measured from the signal of the proton coupled anomeric
carbon, a coupling value which firmly establishing the a-
configuration (Hansen, 1981) of the rhamnose moiety in
1
3
1
3
1
994a). Attachment of the p-hydroxybenzoyl moiety to
17. In the C NMR spectrum, the a-configuration was
derived from the d values of the recorded sugar reso-
nances (Table 2) (Kalinowski et al., 1984). In the HMBC
the glucose carbon C-2 was evidenced by the b-upfield shift
recognized for the signals of the vicinal carbons, C-1 and
C-3 [all in comparison with the chemical shifts of the cor-
responding signals in the spectrum of free a/b glucopyra-
nose] (Nawwar et al., 1984a). In both anomers, C-2 was
found resonating downfield (a-effect) at d 74.7 (C-2a) and
3
spectrum of 17, a J correlation of the anomeric rham-
00
nose proton H-1 (d 5.46) to the aromatic carbon C-4
(d 146.36) allowed positioning of this moiety at this car-
2
bon. The recognizable J correlation of the downfield aro-
7
6.3 (C-2b), thus confirming the final structure of 11 to
matic proton (d 7.69) to the same C-4 carbon was in
accordance with this conclusion. Correlations of the
methoxyl protons (d 4.02) to C-3 (d 141.6) and of the
same downfield aromatic proton (d 7.69) to the same C-
3 carbon confirmed that the site of attachment of the
rhamnosyl moiety is at the C-4 position of the methoxy
ellagic acid moiety. The complete structure of compound
17 was therefore determined to be 3-methoxyellagic acid
4-O-a-L-rhamnopyranoside. This is the first report of a
3-methoxyellagic glycoside bearing its sugar moiety at
the same ring containing the methoxyl function (Kim
et al., 2001; Yazaki and Hillis, 1976; Malhorta and Misra,
1981; Yang et al., 1998).
4
be 2-O-p-hydroxybenzoyl-6-O-galloyl-(a/b)- C -glucopyr-
anose, which represents, to the best of our knowledge, a
new natural product.
1
Compound 17 was obtained as a white amorphous
ꢀ
powder. The molecular ion peak at m/z 461.0712 [MꢀH]
1
13
(
calc.: 461.0715) observed by FTMS and the H, C and
DEPT NMR data suggested the molecular formula
C H O . The characteristic chromatographic properties
2
1
17 12
(
weak mauve spot on PC under UV light) and UV
absorption maxima in methanol suggested the presence of
ellagic acid derivative in 17. The pronounced red shift
of the absorption maxima at 249 and 273 (shoulder) nm of
the aromatic chromophors in the molecule of (17),
observed on addition of NaOAC + H BO (see Section
In addition, the known compounds, gallic acid (1), p-
0
00
00
hydroxybenzoic acid (2), kaempferol 3-O-xylosyl-(1 2 )-
3
3
000 00
3
) might be attributed to the presence of free di-ortho-
glucoside (3), quercetin 3-O-xylosyl-(1 2 )-glucoside (4),
000
00
hydroxyl groups in the aromatic ring(s). Normal acid
hydrolysis of 17 yielded rhamnose (Co-PC), and com-
pound 17a. The latter was also released on incubating
myricetin 3-O-xylosyl-(1 2 )-glucoside (5), 1,6-di-O-gal-
loyl-b-glucose (7), 1-O-p-hydroxybenzoyl-6-O-galloyl-b-
glucose (8), 2,3-di-O-galloyl glucose (9), 2,6-di-O-galloyl
glucose (10), quercetin 3-O-galactoside (12), quercetin 3-
O-glucoside (13), kaempferol 3-O-rhamnoside (14), querce-
tin 3-O-rhamnoside(15), myricetin 3-O-rhamnoside (16),
1
7 at 37 ꢁC for 24 h, together with hesperidinase enzyme
[
a-L-rhamnosidase (Ec 3.2.1.40), lypholized powder from
Penicillium species, Sigma]. Compound 17a was extracted
by EtOAc from the 2 N acidic hydrolysate. This has a
molecular weight of 316 as established by EIMS ([M]
at m/z = 316), corresponding to a molecular formula of
0
3,3 -dimethoxyellagic acid (18), ellagic acid (19), kaempf-
+
erol (20), quercetin (21), and myricetin (22) were also iso-
lated and identified by applying the conventional and
spectral methods of analysis.
C H O . Chromatographic properties (yellowish buff
1
5
8
8
spot on PC under UV light) and UV absorption maxima,
together with the EIMS data suggested that 17a is a
monomethoxy-ellagic acid. Comparison of the 1D NMR
data of 17a with those of free ellagic acid (Nawwar
3. Experimental
0
et al., 1994b), and of 3,3 -dimethoxyellagic acid (Nawwar
3.1. General experimental procedures
et al., 1982) indicated that the methoxyl function in 17a is
attached at C-3. Analysis of the C spectrum of 17a,
1
3
1
H NMR spectra were measured by a Jeol ECA
1
reported here for the first time (see Table 2), was aided
500 MHz NMR spectrometer, at 500 MHz. H chemical

1468
S.A.M. Hussein et al. / Phytochemistry 68 (2007) 1464–1470
1
3
shifts (d) were measured in ppm, relative to TMS and
C
3.4. Extraction, isolation and identification of phenolics from
M. ericifolia leaves
NMR chemical shifts to DMSO-d6 and converted to
TMS scale by adding 39.5. Typical conditions: spectral
1
13
width = 8 kHz for H and 30 kHz for C, 64 K data points
and a flip angle of 45ꢁ. FTMS spectra were measured on a
Finnigan LTQ-FTMS (Thermo Electron, Bremen, Ger-
many) (Department of Chemistry, Humboldt-Universit a¨ t
zu, Berlin). UV recording were made on a Shimadzu
UV–Visible-1601 spectrophotometer. Paper chromato-
graphic analysis was carried out on Whatman No. 1 paper,
The fresh M. ericifolia leaves (5 kg) were homogenized
in EtOH–H O (3:1) mixture (three extractions each with
2
5 L). The dried filtrate (230 g) of the homogenate was
applied to a polyamide 6s (1500 gm) (Riedel-De-H a¨ n
Ag, Seelze Hannover, Germany) column (150 · 7.5 cm)
and eluted with H O followed by H O–MeOH mixtures
2
2
of decreasing polarities to yield seven fractions (I– VII),
which were individually subjected to 2D-PC. Compounds
1 (145 mg) and 2 (125 mg) were isolated pure from frac-
tion I (eluted with 20%) by Column fractionation (CF)
of 3.19 g material over 35 g Sephadex LH-20 using 2 l
using solvent systems: (1) H O; (2) 6 % HOAc; (3) BAW (n-
BuOH–HOAc–H O, 4:1:5, upper layer). Solvent 3 was
2
2
used for PPC.
3
.2. Plant materials
of H O for elution. Compounds 3 (88 mg), 4 (102 mg)
2
and 5 (54 mg) were individually separated from 762 mg
of fraction II (eluted with 30 %) by prep. PC, using
BAW as solvent, while compounds 6 (75 mg), 7 (78 mg),
8 (63 mg), 9 (111 mg) were obtained from 908 mg of frac-
tion III (eluted with 40 %) through precipitation from an
acetone solution by ether (thrice), and the subsequent
prep. PC of the precipitate, using BAW as solvent.
Repeated CF of 2.65 g of fraction IV (eluted with 50%)
Collection of the leaves of M. ericifolia was made at
the Nile river bank near Faculty of Pharmacy, Cairo Uni-
versity, in April 2004. Authentication was performed by
Dr. M. El-Gebali, former researcher of Botany at the
National Research Centre (NRC) of Cairo, Egypt. Vou-
cher specimen was deposited at the herbarium of the
NRC.
on Sephadex LH-20, using n-BuOH saturated with H O
2
3
.3. Preparation of extract for antibacterial assay
yielded pure samples of 10 (122 mg); 11 (79 mg) 12
(134 mg) 13 (102 mg) and 14 (85 mg). Compounds 15
A 20 g sample of the leaves of the plant was homoge-
(134 mg) and 16 (62 mg) were isolated from 775 mg of
fraction V (eluted with 70%) by repeated prep. PC using
BAW as solvent. Compound 17 (185 mg) was obtained
pure by crystallization from MeOH (thrice) of 604 mg
of fraction VI (eluted with 80 %). Compounds 18
(41 mg), 19 (28 mg), 20 (19 mg) 21 (39 mg) and 22
(16 mg) were individually isolated from 792 mg of the last
major column fraction VII (eluted by MeOH) through
repeated prep. PC, using BAW as solvent.
nized in EtO–H O (3:1) (three extractions each with
2
1
00 ml). The dried filtrate of the homogenate was then sub-
jected to antibacterial assay.
3
.3.1. Antibacterial assay
The disc-diffusion method (Cooksey et al., 1993; Sleigh
and Timburg, 1981) was used to determine the inhibition
zones of the extract and compounds 6, 11 and 17 (see
Table 1). The following multiresistant test strains were
used: Staphylococcus aureus Norddeutscher Epidemie-
stamm (multiresistant reference strain, Smith Kline Bee-
cham), S. aureus 34289 (Friedrich L o¨ ffler Institute of
Medical Microbiology, University Greifswald), S. epide-
rmidis 847, S. h a¨ molyticus 535, and Pseudomonas aerugin-
osa 595 (Institute of Hygiene, Greifswald). Paper discs
0
3.4.1. Eugenol 5-O-b-(6 -galloylglucopyranoside), ericifolin
(6)
2
D
5
ꢂ
Off-white amorphous powder, ½aꢁ þ 77:85 (c = 0.14,
MeOH), R -values: 0.33 (H O), 0.48 (HOAc), 0.65
f
2
(BAW). UV max nm in MeOH: 278. HRFIMS: m/z
ꢀ
= 493.1352 [MꢀH] (C H O ). Normal acid hydrolysis
2
3
26 12
(
6 mm diameter) containing the material were deposited
gave glucose, gallic acid and 5-hydroxyeugenol (6a)
(Co-PC). Gallic acid: R -values: 0.33 (H O), 0.55 (HOAc),
0.78 (BAW). UV max nm in MeOH: 272. H NMR: d ppm
6.96s. 5-hydroxyeugenol (6a): R -values: 0.43 (H O), 0.68
on the surface of inoculated agar plates (10 cm diameter)
and incubated for 16 h at 37 ꢁC. Minimal inhibitory con-
centration (MIC) values were determined by standard
serial broth microdilution assay. All test strains were
grown in nutrition medium II (SIFIN) and inoculated
for 16 h at 37 ꢁC.
f
2
1
f
2
(HOAc), 0.82 (BAW). UV max nm in MeOH: 272inflection,
+
1
278,350
. EIMS: [M] : m/z = 180. H NMR (Table
shoulder
1
3
2). C NMR (Table 2). Partial acid hydrolysis yielded 6-
mono-O-galloyl glucose and 5-hydroxyeugenol. 6-mono-
O-galloyl glucose: R -values: 0.67 (H O), 0.75 (HOAc),
0.62 (BAW). UV max nm in MeOH: 273. H NMR: d
ppm: a-glucose moiety: 5.10 (1H, d, J = 3.5 Hz, H-1);
3.50–3.90 (sugar protons overlapped with water protons);
The MIC of the crude extract was found to be
f
2
1
1
. 19.5 lg/ml
against
S.
aureus
(Norddeutscher
Epidemiestamm);
. 39 lg/ml against the strains S. aureus 34289 and S. epi-
dermidis 847;
2
3.92 (1H, m, H-5); 4.38 (1H, d, J = 12.5 Hz, H-6 ); 4.25
a
3
4
. 312 lg/ml against S. haemolyticus 535;
. 1250 lg/ml against P. aeruginosa 595.
(1H, dd, J = 12.5 and 4.5 Hz, H-6 ); b-glucose moiety:
4.60 (1H, d, J = 8 Hz, H-1); 3.50–3.90 (m, sugar protons
b

S.A.M. Hussein et al. / Phytochemistry 68 (2007) 1464–1470
1469
overlapped with water protons); 3.93 (1H, m, H-5); 4.42
HO
OH
(
1H, d, J = 12.5 Hz, H-6 ); 4.30 (1H, dd, J = 12.5 and
a
4
.5 Hz, H-6 ); galloyl moieties: 6.99s, 7.00s. NMR data
2"
b
OH
H
H
1
"
of 6: Table 2.
7
"
'
6"
C
O
H
2
4
O
H
3
glucopyranose (11)
.4.2. 2-O-p-hydroxybenzoyl-6-O-galloyl-(a/b)- C -
1
6
CH
2
H
O
OMe
1
2
D
5
ꢂ
6
Off-white amorphous powder, ½aꢁ ꢀ 44:6 (c = 0.7,
HO
OH
HO
MeOH), R -values: 0.45 (H O), 0.56 (HOAc), 0.41
f
2
O
(
BAW). UV max nm in MeOH: 262. HRFIMS: m/
1
'
ꢀ
OH
z = 451.0877 [MꢀH] (C H O ). Normal acid hydroly-
2
0
19 12
Compound (6): Eugenol-5-O-β-(6'-O-galloylglucopyranoside)
sis gave glucose, gallic acid and p-hydroxybenzoic acid. p-
hydroxybenzoic acid: R -values: 0.54 (H O), 0.72 (HOAc),
f
2
1
0
.88 (BAW). UV max nm in MeOH: 253. H: d ppm 6.8
(
2H, d, J = 8 Hz, H-3 and H-5); 7.75 (2H, d, J = 8 Hz, H-
HO
OH
1
3
2
1
and H-6). C NMR: d ppm 167.5 (C@O), 123.0 (C-1),
32.2 (C-2 and C-6), 115.5 (C-3 and C-5), 161.3 (C-4).
OH
Partial acid hydrolysis yielded 6-mono-O-galloyl glucose
and p-hydroxybenzoic acid (Co-PC). H NMR of 11: d
C
1
O
O
ppm: a-glucose moiety: 5.02 (1H, d, J = 3.5 Hz, H-1);
CH
2
4
.44 (1H, dd, J = 8 and 3.5 Hz, H-2); 3.67 (1H, t,
HO
O
C
J = 8 Hz- H-3); 3.6–3.3 (m, sugar protons overlapped with
water protons); 3.84 (1H, m, H-5); 4.34 (1H, d,
J = 12.5 Hz, H-6 ); 4.15 (1H, dd, J = 12.5 and 4.5 Hz,
HO
OH
a
O
H-6 ); b-glucose moiety: 4.53 (1H, d, J = 8 Hz, H-1);
b
4
.52 (1H, t, J = 8 Hz, H-2); 3.30–3.60 (sugar protons
overlapped with water protons); 3.92 (1H, m, H-5); 4.37
1H, d, J = 12.5 Hz, H-6 ); 4.17 (1H, dd, J = 12.5 and
O
HO
4
1
Compound (11): 2-O-p-hydroxybenzoyl-6-O-galloyl-α/β- C -
(
glucopyranose
a
4
.5 Hz, H-6 ); galloyl moieties: 6.87s, 6.82s; p-hydrox-
b
ybenzoyl moieties: 6.74 and 6.75 (each as a doublet,
J = 8 Hz, H-3 and H-5 in each anomer); 7.69 and 7.70
O
(
each as a doublet, J = 8 Hz, H-2 and H-6 in each ano-
C
O
OMe
1
3
mer). C NMR data of 11: d ppm: a-glucose moiety:
HO
O
8
6
7
9.91 (C-1), 74.7(C-2), 71.2(C-3), 70.9(C-4), 70.8 (C-5),
4.17 (C-6); b-glucose moiety: 95.3 (C-1), 76.3 (C-2),
4.7 (C-3), 71.9 (C-4), 74.4 (C-5), 64.2 (C-6); galloyl moi-
HO
O
C
O
O
eties: 120.8, 119.9 (C-1), 109.26, 109.37 (C-2 and C-6),
46.03 (C-3 and C-5), 138.9, 139.0 (C-4), 166.05, 166.10
C@O); p-hydroxybenzoyl moieties: 120.78, 120.80 (C-1),
Me
HO
1
(
1
OH
HO
Compound (17): 3-methoxyellagic acid 4-O-α-rhamnopyranoside
32.0 (C-2 and C-6), 115.89 (C-3 and C-5), 162.85,
1
62.609 (C@O).
Acknowledgement
3
(
.5. 3-Methoxyellagic acid 4-O-a-rhamnopyranoside
17)
The authors are indebted to AvH (Alexander von Hum-
boldt) foundation for the donation of a Shimadzu UV–Vis-
ible-1601 spectrophotometer to Mahmoud A. M. Nawwar.
White amorphous powder, ½aꢁ ꢀ 14:15ꢂ (c = 0.077,
25
D
MeOH), R -values: 0.23 (H O), 0.38 (HOAc), 0.50
f
2
(
3
BAW). UV max nm in MeOH 249,273shoulder, 335shoulder,
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3
ꢀ
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2
1
25 12
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