Unusual phenolic glycosides from Cotoneaster orbicularis

Article abstract of DOI:10.1016/S0031-9422(99)00598-1

The whole plant of Cotoneaster orbicularis contains the novel di-C- glycosylflavone, 4'',4'''-di-O-β-glucopyranosyl-vicenin II, or 6,8-di-C-β- Cellobiosylapigenin, as well as the hitherto unknown natural phenolic glucoside, gentisic acid 2-O-β-glucopyranoside, or orbicularin. Further phenolics are protocatechuic, anisic, caffeic, p-coumaric acids, catechin, epicatechin, 2''-O-α-rhamnopyranosylvitexin, vitexin, rutin, isoquercetrin, hyperin and naringenin. All structures were determined by routine methods of analysis and confirmed mostly by ¹H- and ¹³C-NMR. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(99)00598-1

Phytochemistry 53 (2000) 699±704
www.elsevier.com/locate/phytochem
Unusual phenolic glycosides from Cotoneaster orbicularis
a
Amani M.D. El-Mousallamy , Sahar A.M. Hussein , Irmgard Merfort , Mahmoud
b
c
d,
A.M. Nawwar *
a
National Research Centre, El-Dokki, Cairo, Egypt
Department of Chemistry, Zagazig University, Zagazig, Egypt
b
c
Institut Fur Pharmazeutische Biologie, Albert-Ludwigs Universitat, D-79104, Freiburg, Germany
National Research Centre, Department of Phytochemistry, Pharmacutical Sciences Division, El-Dokki, Cairo, Egypt
d
Received 14 July 1999; received in revised form 5 November 1999
Abstract
The whole plant of Cotoneaster orbicularis contains the novel di-C-glycosyl¯avone, 40,41-di-O-b-glucopyranosyl-vicenin II, or
,8-di-C-b-Cellobiosylapigenin, as well as the hitherto unknown natural phenolic glucoside, gentisic acid 2-O-b-glucopyranoside,
6
or orbicularin. Further phenolics are protocatechuic, anisic, ca€eic, p-coumaric acids, catechin, epicatechin, 20-O-a-
rhamnopyranosylvitexin, vitexin, rutin, isoquercetrin, hyperin and naringenin. All structures were determined by routine
1
13
methods of analysis and con®rmed mostly by H- and C-NMR. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Cotoneaster orbicularis; Rosaceae; Whole plant; C-Glycosyl¯avones; 6,8-di-C-Cellobiosylapigenin; Phenolic acid-O-glucoside; Gentisic
acid; 2-O-glucopyranoside; Orbicularin; ESI±MS; NMR
1. Introduction
or orbicularin, (2), previously known as a synthetic
product (Boulos, 1983). The known phenolics, gentisic
(
3), protocatechuic (4), anisic (5), ca€eic (6), p-couma-
Extracts of many species in the genus Cotoneaster
Rosaceae) are noted for their cytotoxic, antitumor ac-
tivity; antispasmodic, cardiotonic actions; antiviral and
diuretic properties [Palme, Bilia, Vincenzo & Morelli,
ric (7) acids, catechin (8), epicatechin (9), 20-O-a-rham-
nopyranosylvitexin (10), vitexin (11), rutin (12),
isoquercetrin (13), hyperin (14) and naringenin (15)
were also isolated and identi®ed. It should be noted
however, that C-glycosyl ¯avones (10 and 11) have
previously been found in the genus Cotoneaster
(
1994; Boulos, 1983]. Among these species, Cotoneaster
orbicularis has never been subjected to phytochemical
investigation. The plant is a thorny evergreen shrub
with small leaves and red fruits. It grows wild in the
southern part of the Sainai peninsula. In the present
paper the phytochemical investigation of Cotoneaster
orbicularis phenolics from an aqueous alcoholic extract
of the whole plant is described. This investigation
resulted in the isolation and characterisation of the
new unusual di-C-glycosylapigenin, 6,8-di-C-cellobiosy-
(
Palme, Bilia, Vincenzo & Morelli, 1994).
The analytical data of (1) was compared with that
of the known positional isomer, 6,8-di-C-sophorosyla-
pigenin, isolated once before from Ephedra aphylla
(
Hussein, Barakat, Nawwar & Willuhn, 1997). The
new cellobiosylapigenin is interesting because it con-
tains the unusual interglucosidic linkage (1 4 4). This
linkage has been determined before in two ¯avone O-
glucosylglucosides characterized from Salvia uliginosa
lapigenin (1) as well as the unknown natural phenolic,
4
2
,5-dihydroxybenzoic acid-2-O-b- C -glucopyranoside,
1
(
Veitch, Grayer, Irwin & Takeda, 1998) and in an O-
glucosyl-C-glucosyl¯avone, namely, 40-O-glucosylswer-
tisin (zivulgarin) from Zizyphus spinosus (Jay, 1986) as
well.
*
Corresponding author.
E-mail address: nawwar@worldnet.com.eg (M.A.M. Nawwar).
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00598-1

7
00
A.M.D. El-Mousallamy et al. / Phytochemistry 53 (2000) 699±704
It should be mentioned also, that gentisic acid glyco-
eties. Complete acid hydrolysis (methanolic 2 M HCl,
7 h) of (1) gave 6,8-di-C-glucosyl apigenin, or vicenin
II (CoPC, UV, H- and C-NMR analysis) and glu-
cose (CoPC). The former also was released from (1)
on b-glucosidase enzymic hydrolysis. Consequently (1)
is proved to be a di-O-b-glucosylvicenin II, which pos-
sides have recently been identi®ed as derivatives of sal-
isylic acid, a compound now known to be synthesised
in many plants during defence responces. The acid has
been encountered in nature only as its 5-O-glucoside
(Fujii, Aoki, Komoto & Munakata, 1971; Zane &
Wender, 1964), but not as the 2-O-glucoside.
1
13
sessed quite distinct R -values (Table 1) from that of
f
its positional isomer, 6,8-di-C-sophorosylapigenin, iso-
lated previously from Ephedra aphylla (Hussein, Bara-
kat, Nawwar & Willuhn, 1997).
2. Results and discussion
1H-NMR spectral analysis of (1) failed to unravel
the ambiguity about the site of attachment of the two
O-glucoside moieties to vicenin II in the molecule of
(1). The measured spectrum (room temperature,
2
D-PC screening of the aqueous ethanolic (1:3)
whole Cotoneaster orbicularis plant extract revealed an
oligoglycosylated ¯avonoid [(1), dark purple spot on
PC under UV light, turned light yellow when fumed
DMSO-d ) was closely similar to that of the positional
6
with ammonia, with high R -values in aqueous sol-
f
isomer 6,8-di-C-sophorosylapigenin. It revealed four b-
anomeric glucose proton resonances, all appearing as
doublets (J = 8 Hz) at d ppm 4.95, 4.85, 4.75 and
4.72. In addition, a broad multiplet, located in the
region from 3.22 to 3.85 ppm, was assigned to the
remaining 24 sugar proton resonances of the four glu-
cosyl moieties and to the hydroxyl and water protons
as well. In this spectrum the typical pattern, of proton
resonances which characterises 6,8-disubstituted api-
genin was also recognised ‰d ppm 6.6 (s, H-3), 6.8 (d, J
= 8 Hz, H-30 and H-50), 7.9 (d, J = 8 Hz, H-20 and
H-60)].
vents and low mobility in organic ones], together with
a complicated mixture of phenolic, phenylpropenoid
acids (characterisitic ¯uorescence under short and long
UV light on PC) and ¯avonoid glycosides (dark purple
spots on PC under UV light, changing to yellow or
orange colour when fumed with ammonia). All com-
pounds (1±15) were isolated and identi®ed by polya-
mide CC followed by applying a combination of
Sephadex LH-20 column fractionation and preparative
PC. Two of the separated compounds (1 and 2) are
new. The remaining compounds (3±14) are known and
gave chromatographic, UV spectral and hydrolytic
data identical with those of gentisic, protocatechuic,
Precise determination of the site of attachment of
the two O-glucosyl moieties to vicenin II in the mol-
13
anisic, ca€eic, p-coumaric acids, catechin, epicatechin,
1
ecule of (1) was then achieved through C-NMR,
13
vitexin-20-a-O- C -rhamnopyranoside, vitexin, rutin,
including HMBC spectral analysis. The
C-NMR
4
isoquercetin, hyperin and naringenin, respectively. The
structures of these known compounds were con®rmed
spectrum revealed two O-b anomeric glucosyl carbon
resonances at d ppm 103.3 and 103.9, comparable to
103.3 ppm showed by the b-anomeric O-glucosyl car-
bon in free cellobiose (Breitmayer & Voelter, 1978).
The spectrum also exhibited a total of 18 distinct
sugar carbon resonances in the region from d ppm
60.0 to 82.0. The resonances at d ppm 60, 60.1, 61.0
and 61.3 were obviously due to the four unsubstituted
methylenic glucose carbons in the four sugar moieties
of (1), while resonances at 77.0, 77.1, 69.1, 69.8, 77.8
78.1 are attributable to C32, C-32', C-42, C-42', C-
52 and C-52' in two terminal O-glucosyl moieties,
attached to the 6-C and 8-C primary glucosyl moieties,
1
13
by H- and C-NMR spectral analysis.
Compound (1), was isolated as a yellowish white
amorphous powder which possessed a molecular ion of
�
[
M-H] m/z = 917 in negative ESI±MS, correspond-
ing to a M of 918 amu. Chromatographic properties
r
and UV spectral analysis (see Table 1) in methanol
and in the presence of diagnostic shift reagents (Har-
borne & Williams, 1975; Mabry, Markham & Tho-
mous, 1969) together with the results of the ESI±MS
analysis suggested for (1) an oligo-C-hexosylapigenin
structure whose molecule contains four hexose moi-
Table 1
Chromatographic and UV data of compounds 1 and 2
Compound
Chromatographic properties ꢀRf sꢀÂ100†
UV spectral data lmax (nm)
H
2
O
HOAc
BAW
MeOH
NaOAc
NaOAc±H
3
BO
3
AlCl
3
MeONa
1
32
23
35
68
45
55
48
70
17
33
19
78
272, 332
272, 333
236, 315
239, 334
283, 390
282, 393
284, 391
283, 390
282, 300, 345, 382
280, 305, 345, 383
275, 360, 402
275, 361, 402
238, 326
Vicenin-II
2
Gentisic acid

A.M.D. El-Mousallamy et al. / Phytochemistry 53 (2000) 699±704
701
respectively. Besides these, two other carbon reson-
ances have been recognised, in this spectrum at d ppm
NMR spectral analysis] together with glucose (coPC).
The former was released from (2) on b-glucosidase
enzymic hydrolysis. To ®nd out how the gentisic acid
and glucose moieties are incorporated in the molecule
of (2) NMR spectral analysis were carried out. The
7
2
3.4 and 74.1 and were assigned to the C-22 and C-
2' in the two terminal glucosyl moieties.
Comparison of the chemical shifts of the primary
1
glucose carbon resonances in b-glucosyl (1 4 4) -b-glu-
cose, (b-cellobiose) and those of free b-glucose itself
led to the deduction of substituent additive rules which
when applied to the chemical shifts of the carbon reso-
naces of the 6-C and 8-C-glucosyl moieties in vicenin
II (Nawwar, El-Mousallamy, Barakat, Buddrus &
linscheid, 1989), would give calculated chemical shifts
which could be compared with those recorded for the
two primary 6-C and 8-C-glucosyl moieties of com-
pound (1). Both calculated and recorded values were
in close agreement, thus proving that resonances in the
recorded spectrum located at d ppm 72.8, 73.3,
H-NMR spectrum (room temperature. DMSO-d6)
revealed, in addition to the characteristic proton reson-
ances pattern of gentisic acid moiety ‰d ppm 7,2 (d, J
= 2 Hz, H-6'), 6.9 (dd, J = 2 Hz and J = 7.5 Hz, H-
4', 6.85 (d, J = 7.5 Hz, H-3')] another di€erent pat-
tern of proton resonances belonging to a b-glucose
moiety ‰d ppm 4.7 (d, J = 8 Hz, H-1), 3.2-3.85 (m, six
glucose protons hidden by hydroxyl and water pro-
tons]. Glucosidation at C-2' of the gentisic acid moiety
was evidenced by the low®eld shift of H-3' proton res-
onance ꢀDd ˆ 0:2 ppm) when compared with the corre-
sponding signal in the spectrum of free gentisic acid.
The value of the coupling constant of the anomeric
glucose proton resonance (J = 8 Hz) indicated that
70.6,71.0, 77.5, 78.9, 81.5, 82.0, 80.9 and 81.0 are
assignable to C-10, C-11, C-20, C-21, C-30, C-31, C-40,
C-41, C-50 and C-51, respectively (taking into account
that chemical shifts with the same number, but with
di€erent superscript may be interchanged). Furthe-
more, the absence of any sugar carbon resonances
with chemical shift lower than 82.0 ppm except those
of the O-b-anomeric carbons would directly eliminate
the possibility of the presence of a laminaribiosyl (glu-
cosyl 1 4 3 glucose) moiety in the molecule of (1),
4
the sugar moiety adapts a C1 conformation and a b-
con®guration (Nawwar, Ishak, Michael & Buddrus,
1984). The weight of evidences given above proved
4
that (2) is gentisic acid 2-O-b- C1-glucoside. Con®r-
1
3
mation of this structure was then achieved through
C
1
13
13
and H± C coupled NMR analysis. The C spectrum
contained, as expected a total of 13 distinct carbon res-
onances. Assignments were aided by comparison with
1
3
(
Karl, Pederesen & Mueller, 1980). The two C-glucosyl
the C-NMR data reported for similar phenolic O-
glucosides (e.g. ¯avones 5-O-glucosides and gentisic
acid itself) (Merkham & Chari, 1982). The b-anomeric
glucose carbon resonance was readily identi®ed from
its characteristic d value at 102 ppm, while the most
up®eld glucose carbon resonance, in this spectrum,
located at d 60.7, ppm was assigned to the methylenic
carbon. Other resonances in the sugar region exhibited
d-values which were in accordance with those reported
for glucose in previously reported phenolic O-gluco-
sides (Merkham & Chari, 1982; Nawwar, Buddrus &
moieties linked to the ¯avonoid carbons C-6 (at 107.5
ppm) and C-8 (at 105.3 ppm) were unambiguously
con®rmed, in an HMBC spectrum, by a long-rang cor-
2
relation ꢀ J† between their anomeric protons (H-10 at
4
.95 ppm and H-11 at 4.85 ppm) and these ¯avonoid
carbons. This latter spectrum also allowed the inter-
3
connectivity, across three bonds ꢀ J), of the remaining
anomeric glucose protons, H-12 at 4.75 ppm and 12'
at 4.72 ppm with the primary glucose carbons, C-40
and C-41 at 81.5 and 82.0 ppm.
1
13
Consequently, compound (1) is identi®ed to be
Bauer, 1982). The H± C coupled spectrum of (2)
showed, in the aromatic region, one doublet for the
gentisic acid carbons C-1' (J = 7 Hz), one doublet for
C-6' (J = 162 Hz), a double doublet for C-3' (J =
158.6 Hz and J = 6.5 Hz) and another double doublet
for C-4' (J = 161 Hz, J = 6.5 Hz) at d ppm 115.3,
117.7, 117.4 and 124.5, respectively. It also showed a
doublet (J = 7.Hz) for carbons C-2', a clear triplet (J
= 6.5 Hz) for C-5' and a doublet (J = 6 Hz) for C-7'
at d ppm 147.9, 156.4 and 171.6, respectively. Com-
parison of these chemical shift values with those
recorded for gentisic acid (room temperature DMSO-
d6), [112.9 (C-1), 149.6 (C-2), 114.9 (C-3), 124.1 (C-4),
154.4 (C-5), 118.1 (C-6) and 172.0 (C-7)] showed an
up®eld shift, Dd ˆ 1:73 ppm for the C-2' resonance ac-
companied by down®eld shifts of Dd ˆ 4:4 and 2.5
ppm for the resonances of the ortho carbons C-1' and
C-3', respectively. These changes in chemical shifts are
4
0,41-di-O-b-glucopyranosylvicenin II, or 6,8-di-C-cel-
lobiosylapigenin, which represents to the best of our
knowledge, a new natural product.
The new compound (2) was obtained as an amor-
phous o€-white powder which possesses phenolic char-
acters (dirty blue colour with FeCl ) and UV spectral
maxima: 236, 315 nm in MeOH and 238, 326 nm in
MeOH + MeONa. Negative FAB mass spectral
analysis established that (2) was a dihydroxybenzoic
3
�
acid hexoside ([M-H] at m=z ˆ 315† of a M = 316
r
amu. This could also be deduced from the EI-M spec-
trum of the compound which revealed a fragment ions
at m/z: 154, 136 and 107 consistent with a dihydroxy-
benzoic acid as the aglycone moiety. On normal acid
hydrolysis (2 N aqueous HCl, 3 h, 1008C) compound
(
[
2) yielded 2,5-dihydroxybenzoic acid, gentisic acid
13
coPC, UV (Table 1), 1H, C and 1H± C coupled
13

7
02
A.M.D. El-Mousallamy et al. / Phytochemistry 53 (2000) 699±704
obviously due to the introduction of a glucose moiety
at position 2' of the gentisic acid moiety to form the
molecule of (2), (see Formula).
used for prep. PC on Whatman no. 3 MM paper and
solvents 3 and 4 for sugar analysis.
4
Conformation of the glucose moiety as C1 followed
3.1. Plant material
from the b-con®guration discussed above. Also, the
chemical shift values of the resonances of all glucose
carbons con®rmed the pyranose form of this sugar
moiety. Consequently, compound (2) is ®nally ident-
i®ed to be gentisic acid 2-O-b- C1-glucopyranoside, a
compound which is known as a synthetic product
Fresh shrubs of Cotoneaster orbicularis Schlecht.
were collected from the mountains of south Sinai
around Saint Catharine, Egypt, in November and
authenticated by Dr. E. El-Garf, Teacher of Botany,
Faculty of Science, Cairo University. A voucher speci-
men is deposited in the Herbarium of The NRC.
4
(Wagner, 1958), but which has not been reported, pre-
viously as a natural product.
3
.2. Isolation and identi®cation
Shrubs of C. orbicularis, dried in the shade in an
air-draft, were comminuted to powder (2 kg) and
exhaustively extracted with EtOH/H O (3:1). The
2
extract, dried in vacuo (204 g) was analysed by 2 D-
PC. A 29.5 g portion of the extract, dissolved in 50 ml
MeOH was applied to a polyamide 6S CC (Riedel-De
Haen AG, Seelze Hannover, Germany) and eluted by
H O followed by H O/EtOH mixtures of decreasing
2
2
polarities to a€ord 10 major frs (I-X).
Compounds 1 (103 mg) and 2 (92 mg) were isolated
from fr. I by repeated CC over Sephadex LH-20, using
H O and n-BuOH saturated with H O for elution.
2
2
Prep. PC, using BAW as solvent system, yielded pure
sample of each. Compound 3 (76 mg), 4 (88 mg) and 5
(
57 mg), were separated from fr. III by repeated CC
on Sephadex LH-20, using 95% EtOH as an eluent.
Compound 6 (102 mg) and 7 (86 mg) were isolated
from fr. IV by CC fractionation on polyamide, using a
mixture of MeOH/C H CH /H O (60:38:2) as an elu-
6
5
3
2
ent and compounds 8 (144 mg) and 9 (78 mg) from fr.
V, by CC fractionation over Sephadex LH-20, using
EtOH/H O (1:2) for elution. Compounds 10 (122 mg),
2
11 (106 mg) and 12 (152 mg) were obtained from fr.
VI through CC over Sephadex LH-20, using n-BuOH
saturated with H O as solvent. Compounds 13 (87 mg)
2
and 14 (52 mg) were separated from fr. VII through
prep. PC, using n-BuOH saturated with H O as sol-
2
vent and compound 15 (85 mg) were isolated from fr.
VIII through polyamide column fractionation using
3. Experimental
EtOAc saturated with H O as solvent for elution.
2
1H-NMR 270 MHz. H-NMR resonances were
1
3.3. 6,8-di-C-cellobiosylapigenin (1)
13
measured in DMSO-d , relative to TMS and C-
6
NMR were converted to TMS scale by adding 39.5.
1
R s, R and UV spectral data: Table 1. M : 918,
r
ESI±MS: negative ion: m/z (rel. inten.): 917.4(55), [M-
f
f
Typical conditions: spectral width = 4000 for H,
1
3
�
�
1
4
3,000 Hz for C, 32 K data points and a ¯ip angle of
58. Negative ESIÐM spectra were measured on SSQ
H] , 593(100), [vicenin II-H] . 1 was hydrolysed with
2 M aqueous methanolic HCl (1:1) at 1008C for 7 h to
give vicenin II and glucose. The former was precipi-
tated from the cold aqueous hydrolysate after removal
of MeOH. Vicenin II: Rf values and UV data: Table 1;
®
nnigan MAT 4600 mass spectrometer. PC was carried
out on Whatman no. 1 paper, using solvent systems:
1) H O; (2) 15% HOAc±H O; (3) BAW (n-BuOH±
(
2
2
1
HOAc±H O, 4:1:5, top layer); (4) C H -n-BuOH±
2
H-NMR: aglucone moiety: d ppm 6.76 (s, H-3), 6.92
6
6
H O-pyridine, 1:5:3:3, top layer). Solvents 1±3 were
2
(d, J = 8 Hz, H-3' and H-5'), 8.0 (d, J = 8 Hz, H-2'

A.M.D. El-Mousallamy et al. / Phytochemistry 53 (2000) 699±704
703
Table 2
1
3
C-NMR chemical shifts (ppm) of the new Cotoneaster orbicularis and the related compounds
a
Carbon
40,41-di-O-glucosyl-vicenin II
Vicenin II
Gentisic acid 2-O-b-glucoside
Gentisic acid
1
2
3
4
5
6
7
8
9
1
1
2
3
4
5
6
7
1
2
3
4
5
6
1
2
3
4
5
6
102.0
73.3
76.5
69.7
77.1
60.7
164.2
103.9
182.4
158.6
107.5
161.3
105.3
155.1
103.9
121.5
129.1
115.9
160.8
115.9
129.1
164.1
102.6
182.3
158.5
107.5
161.2
105.3
155.1
103.8
121.5
129.0
115.8
160.7
115.8
129.0
0
'
'
'
'
'
'
'
115.3
147.9
117.4
124.5
156.4
117.7
171.6
112.9
149.6
114.9
124.1
154.4
118.1
172.0
0 and 11
0 and 21
0 and 31
0 and 41
0 and 51
0 and 61
2 and 12'
2 and 22'
2 and 32'
2 and 42'
2 and 52'
2 and 62'
72.8 and 73.3
70.6 and 71.0
77.5 and 78.9
81.5 and 82.0
80.9 and 81.0
61.0 and 61.3
103.6 and 103.2
73.4 and 74.1
77.0 and 77.1
69.1 and 69.8
77.8 and 78.1
60.0 and 60.1
74.0 and 73.3
71.9 and 70.8
78.8 and 77.8
70.5 and 69.1
81.8 and 80.8
60.5 and 61.3
a
Chemical shifts of carbonc with the same number but with di€erent superscript may be interchanged.
and H-6'); sugar moieties: 4.84 (broad s, Dn1=2 ˆ 16
Hz, H-1' and H-11), 3.08±3.88 (m, 12 sugar protons
hidden by hydroxyl and water proton resonances). Hy-
drolysis of 1 with b-glucosidase (Nawwar, Hussein &
Merfort, 1994), (from sweet almond meal, Merck)
yielded vicenin II (coPC). 1H-NMR of 1: aglucone
moiety: 6.6 (s, H-3), 6.8 (d, J = 8 Hz, H-3' and H-5'),
and gentisic acid. The latter was extracted by EtOAc.
1
Gentisic acid: R s and UV data: Table 1; H-NMR in
f
DMSO-d : d ppm 6.75 (d, J = 7.5 Hz, H-3), 6.95 (dd,
6
J = 7.5 and J = 2.5 Hz, H-4), 7.15 (d, J = 2.5 Hz,
H-6); C-NMR: Table 2. b-glucosidase enzymic hy-
drolysis of 2 yielded gentisic acid (coPC). H-NMR of
13
1
2 in DMSO-d : gentisic acid moiety: d ppm 6.85 (d,
6
7
.9 (d, J = 8 Hz, H-2' and H-6'); sugar moieties: d
ppm 4.95, 4.85, 4.75 and 4.72 each as d, J = 8 Hz, H-
0, H-11, H-12 and H-12'; 3.22±4.2 (broad m, 24
sugar protons hidden by hydroxyl and water protons).
J = 7.5 Hz, 3'), 6.9 (dd, J = 7.5 and J = 2.5 Hz,
H-4'), 7.2 (d, J = 2.5 Hz, H-6'); glucose moiety; 4.7
(d, J = 8 Hz, H-1), 3.2±3.85 (broad m, 6 sugar proton
resonances hidden by sugar hydroxy proton and
1
1
13
13
HMBC: Connectivities of most H and C sugar res-
onances were determined, because of signals ovelap.
water proton resonances). C-NMR of 2: Table 2.
13
C-NMR of 1: Table 1.
Acknowledgements
4
3
.4. 2,5-Dihydroxybenzoic acid 2-O-b- C -
1
This work was excuted through project no. W-383-/
6-II of Deutsche Forschungsgemeinschaft for which
the authors feel very grateful.
4
glucopyranose, gentisic acid 2-O-b- C -glucopyranose,
1
orbicularin (2)
Rfs: Table 1. UV data: Table 1. Mr of 1, 316, nega-
�
tive ESI±MS, [M-H] : m/z (rel. intent): 315 (48). Frag-
ment ions in EI±MS, m/z (rel. inten.): 154 (76), 136
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