Acylated flavonol diglucosides from Lotus polyphyllos

Article abstract of DOI:10.1016/S0031-9422(02)00177-2

Three acylated flavonol diglucosides, kaempferol 3-O-β -(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; quercetin 3- O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; isorhamnetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside were isolated from the

Full text of DOI:10.1016/S0031-9422(02)00177-2

Phytochemistry 60 (2002) 807–811
www.elsevier.com/locate/phytochem
Acylated flavonol diglucosides from Lotus polyphyllos
Amani M.D. El Mousallamia, Manal S. Afifib, Sahar A.M. Husseinb,*
aDepartment of Chemistry, Zagazig University, Zagazig, Egypt
bNational Research Center, Dokki, Cairo, Egypt
Received in revised form 15 January 2002; accepted 15 May 2002
Abstract
Three acylated flavonol diglucosides, kaempferol 3-O-b-(600-O-E-p-coumaroylglucoside)-7-O-b-glucoside; quercetin 3-O-b-(600-O-
E-p-coumaroylglucoside)-7-O-b-glucoside; isorhamnetin 3-O-b-(600-O-E-p-coumaroylglucoside)-7-O-b-glucoside were isolated from
the whole plant aqueous alcohol extract of Lotus polyphyllos. The known 3,7-di-O-glucosides of the aglycones kaempferol, quer-
cetin and isorhamnetin were also characterized. All structures were established on the basis of chemical and spectral evidence.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Lotus polyphyllos; Leguminosae; Acylated flavonol diglucosides; Kaempferol 3-O-b-(600-O-p-coumaroylglucoside)-7-O-b-glucoside;
Quercetin 3-O-b-(600-O-p-coumaroylglucoside)-7-O-b-Glucoside; Isorhamnetin 3-O-b-(600-O-p-coumaroylglucoside)-7-O-b-glucoside
1. Introduction
region of the Mediterranean coastal strip of Egypt. In the
present communication, we describe the isolation and
structure elucidation of the three new acylated flavonol
glycoside (4–6), from the aqueous alcoholic whole plant
extract of Lotus polyphyllos E. D. Clarke (Syn. Lotus
argenteus Webb & Berthel). In addition, the known
compounds, kaempferol 3,7-di-O-glucoside (1), querce-
tin 3,7-di-O-glucoside (2) and isorhamnetin 3,7-di-O-
glucoside (3) were characterized from the same extract.
Also, the 13C NMR data of the known compounds (2
and 3) are recorded and assigned here for the first time.
The genus Lotus (Leguminosae) includes both acya-
nogenic and high cyanogenic species (Rizk, 1986). Some
of these species are used as a source of lectins. In folk
medicine, they are used as contraceptive, prophylactic
agents and for treating sexually transmitted disorders,
for oral alimentary and for peptic ulcer (Oldham et al.,
1995; Oldham and Krivan, 1996; Hirata et al., 1998).
Previous phytochemical investigations have proved the
isolation of the 3-O-b-galactosides of the flavonol gos-
sypetin, its 7- and 8- methyl ethers from L. corniculatus
(Harborne, 1969; Nielsen, 1970; Jay et al., 1978) and the
isolation of kaempferol 3-O-b-glucoside together with its
7-O-a-rhamnoside derivative from L. tenuis (birds tre-
foil), (Strittmater et al., 1992). Recently, L. hebranicus
Hochst ex. Brand was reported to contain kaempferol
7-O-a-rhamnoside together with its 3-O-a-rhamnoside
and 3-O-sophoroside derivatives. Also kaempferol 3-O-
a- rhamnoside-7-O-sophoroside and isorhamnetin 3-O-
b-glucoside-7-O-b-glucoside have been reported from
the same plant (Kassem, 2001). However, there are no
reports on the constitutive flavonoids of Lotus poly-
phyllos which grows wild in sand dunes of the western
2. Results and discussion
The concentrated 70% aqueous ethanol extract from
a homogenate of the dried whole plant was fractionated
by column chromatography over Sephadex LH 20,
using water/methanol mixtures of decreasing polarities.
Repeated preparative paper chromatography of the 30
and 50% aqueous methanol column fractions afforded
pure samples of compounds (1–6). The known com-
pounds (1–3) showed chromatographic, UV absorption
and hydrolytic data identical with those reported for
kaempferol 3,7-di-O-glucosides (Egger, 1961), quercetin
3,7-di-O-glucoside (Harborne, 1982) and isorhamnetin
3,7-di-O-glucoside (Krishnamurti et al., 1965), respec-
tively. The 13C NMR (see Table 2) spectral data of
* Corresponding author. Fax: +20-233-70931.
E-mail address: nawwar@worldnet.com.eg (S.A.M. Hussein).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00177-2

808
A.M. D. El Mousallami et al. / Phytochemistry 60 (2002) 807–811
compounds 2 and 3 were recorded and assigned here for
the first time.
3-O - b - (600 - p - coumaroylglucoside) (chromatographic
properties and H NMR), (Kumar et al., 1985). Conse-
1
quently, 4 is kaempferol 3-O-(p-coumaroyl)glucoside)-7-
O-glucoside. The result of negative ESI–MS analysis of
4 supported this, indicating a molecular ion peak at [M–
H]ꢁ 755, corresponding to a molecular weight (Mr) of
756. In order to determine unambiguously the structure
of 4, specially the site of attachment of the p-coumaroyl
moiety to the 3-O-glucoside moiety, it was necessary to
1
apply NMR spectroscopic analysis. The H NMR spec-
trum of 4 (DMSO-d6, room temperature) revealed two
distinct anomeric hexose proton resonances at ꢀ ppm 5.02
and 5.55 (each d, J=8.5 Hz) attributed to the anomeric
glucoside moieties at the 7- and 3-positions in 4.
The spectrum also showed a pair of glucose proton
resonances at ꢀ 4.26 (dd, J=12 and 4.5 Hz) and 4.12
(m), assignable to the two methylenic glucose protons
whose hydroxyl group is acylated by the p-coumaroyl
moiety. Resonances of the two methylenic protons in
the second glucose moiety, together with all of the
remaining glucose protons appeared more upfield in the
region from ꢀ 3.3 to 3.9, overlapped with water protons
signal. In this spectrum the characteristic protons of the
E-p-coumaroyl moiety resonated at ꢀ 6.15 (d, J=16 Hz,
H-80000), 6.8 (d, J=8 Hz, H-30000 and H-50000),7.55 (d, J=16
Hz, H-70000), 7.6 (d, J=8 Hz, H-20000 and H-60000). The
spectrum showed in addition, the presence of a 7-O-
substituted kaempferol moiety by the proton resonances
at ꢀ 6.32 (d, J=2.5 Hz) and 6.72 (d, J=2.5 Hz), assign-
able to the H-6 and H-8 of this moiety. These two che-
mical shift values were closely similar to those reported
for the corresponding proton resonances in the spectrum
of kaempferol-3,7-di-O-glucoside 1 (Markham and Gei-
ger, 1994) In this spectrum the chemical shifts of the B ring
proton resonances (see Experimental) were found to be in
close agreement with the proposed structure.
Compound 4, a yellow amorphous powder, showed
chromatographic properties ( dark purple spot on paper
chromatogram under UV light, turning lemon yellow
when fumed with ammonia vapour, moderate migration
in aqueous and organic solvents) and colour reactions (a
lemon yellow colour with Naturstoffe and a bright-yellow
colour with 2% ZrOCl2 which disappeared on addition
of 2% citric acid and water), (Liu et al., 1997) char-
acteristic of a 40-oxygenated flavonol bearing a free
hydroxyl at the 5-position and a substituted one at the
3-position. UV spectral analysis (see Table 1) in metha-
nol and on addition of shift reagents (Harborne and
Williams, 1975; Mabry et al., 1969) confirmed the pre-
sence of a free hydroxyl at 40-position (stable MeONa
spectrum) and a substituted hydroxyl at the 7-position
(no shift with NaOAc). Normal acid hydrolysis of 1(2
The 13C NMR analysis confirmed the structure of 4.
As expected, the spectrum (DMSO-d6, room tempera-
ture) exhibited twelve distinct glucose carbon resonances
(see Table 2). The two b-anomers were recognized from
the downfield resonances at ꢀ 99.8 and 100.6, while the
most upfield resonances at ꢀ 60.1and 63.0 were assigned
to the methylenic C-6000 glucose carbon bearing a free
N aqueous HCl, 1h, 010
ꢀC) yielded glucose (com-
parative paper chromatography Co-PC), kaempferol
and p-coumaric acid (Co-PC, UV spectral and 1H
NMR). On b-glucosidase enzymatic hydrolysis (incu-
bation together with 0.5 ml of the enzyme in acetate
buffer, pH 5.1at 37 ꢀC, for 24 h) 4 yielded kaempferol
Table 1
Chromatographic and UV data of the new flavonoids (4–6)
Compound
Chromatographic
properties (Rfsꢂ100)
UV spectral data lmax mn
H2O HOAc 15% BAW MeOH
NaOAc (a)
(a) +H3BO3
268, 318
AlCl3
MeONa
4
5
6
52
50
48
58
55
52
42
44
40
43
57
268, 316, 360a
268, 317, 366
275, 312, 392 277, 362
256, 265a, 297a, 313, 354a 256a, 265, 300, 297a, 312, 374 273, 299, 313, 438a 243a, 274, 367
253, 266a, 300a, 313, 354 255, 266, 302a, 313, 376a
269, 295a, 312, 356a 278, 302a, 313, 366a
257, 265a, 314, 355 267, 314a, 398 269, 365
277, 300a, 312, 405 277, 312, 379
Kaempferol-3-O-b- 28
(600-p- coumaroyl)-
glucoside
a
Inflection.

A.M. D. El Mousallami et al. / Phytochemistry 60 (2002) 807–811
809
Table 2
13C-NMR chemical shifts (ppm) of the flavonoid (2–6) of Lotus poly-
phyllos
were accompanied by downfield shifts of the resonances
of the corresponding o- and p-carbons all in comparison
with the corresponding resonances in the spectrum of
the aglycone, kaempferol (see Experimental), (Mark-
ham et al., 1978). Furthermore, the measured chemical
shift values of the carbon resonances of the two glucose
moieties confirmed that the sugar cores exist in the
pyranose form. Consequently, 4 is identified as kaempferol
3-O-b-(600 -p-coumaroylglucopyranoside)-7-O-b-gluco-
pyranoside, which has not been reported previously in
nature.
Carbon
2
4
3
5
6
2
156
156.9
133.4
177.6
160.8
99.3
156.1
133.5
177.5
160.6
99.5
156.5
133.1
177.6
160.8
99.2
156.2
133.2
177.5
160.3
99.4
3
133.6
177.6
160.9
99.3
4
5
6
7
162.8
94.3
156
162.8
94.4
163.2
94.6
162.4
94.2
156
162.9
94.5
157
8
9
155.9
105.5
120.9
130.3
115.9
159.8
115.9
130.3
100.6
156.9
105.8
122.2
113
Compound 5 was isolated as faint yellow amorphous
powder which appeared dull purple on chromatograms
under UV light, turning orange when fumed with
ammonia vapour. It gave glucose, quercetin and p-cou-
maric acid (CoPC), on normal acid hydrolysis. Com-
pound 5 exhibited a molecular weight (Mr) of 772 in
negative ESI–MS ([M–H]ꢁ 771). These data together
with Rf values and UV spectral analysis (see Table 1),
10
10
105.7
121
105.6
122.1
115.3
144.8
148.8
115.9
121
105.8
122.4
20
115.4
144.7
148.6
116.3
121.6
100.5
113,5
149.6
147
30
149.5
146.9
115.9
122.8
105.8
74.1 74.3
77.7
40
500
115.7
600
100
200
300
400
500
6000
1000
2000
3000
4000
5
122,0
100.7
106.8
74.174.174.2
77.6
69.9
76.4
60.9
99.6
77.2
77.5
69.9
74.4
63.7
99.7
73.2
76.3
69.6
76.9
77.6
69.9
74.3
63.3
99.8
1
indicated that 5 is the quercetin analogue of 4. H and
69.8
74.5
63.5
99.9
69.9
76.5
13C NMR (see Table 2) spectroscopic analysis of 5 con-
firmed its structure to be quercetin 3-O-b-(600-E-p-cou-
maroylglucopyranoside)-7-O-b-glucopyranoside which
has not been reported before as a natural product.
The new compound 6 (dull yellow amorphous pow-
der) was identified as the isorhamnetin analogue of 4
and 5 from chromatographic, UV spectral (see Table 1),
hydrolytic, and negative ESI–MS ([M–H]- 785, corre-
sponding to molecular weight (Mr) of 786) data. Its
structure was confirmed by 1H and 13C NMR (see
Table 2) analysis to be isorhamnetin 3-O-b-(600-E-p-
coumaroylglucopyranoside) - 7 - O - b - glucopyranoside,
which represnts the third new natural product.
60.6
99.7
73.173.173.2
73
76.4
69.5
76.5
60.6
76.4
76.6
69.8
76.5
70
69.7
76.4
76.8
76,5
6000
10000
20000
30000
40000
50000
60000
70000
80000
90000
Ome
60.160.3
124.6
130
60,4
60.5
124,8
130.3
124.8
130.2
116
115.9
159.9
115.9
130
116.1
159.9
116.1
130.3
144.3
115
159.8
116
130.2
144.2
116
144
114.7
166.1
It should be noted that this is the first reported occur-
rence of acylated flavonol glycosides in Lotus species.
165.9
166
55.9
55.8
3. Experimental
hydroxyl (located at the 7-position of the kaempferol
moiety) and to the methylenic C-600 (at the 3-position),
respectively.
NMR: Jeol EX-270 spectrometer, 270 MHz (1H NMR)
1
and 67.5 MHz (13C NMR), respectively. H resonances
were measured relative to TMS and 13C NMR resonances
to DMSO-d6 and converted to TMS scale by adding 39.5.
ESI–MS: Micromass Quattro-LC triple quadrupole mass
spectrometer equipped with a ‘‘Z-Spray’’ electrospray ion
source. PC (descending): Whatman no. 1paper, using
solvent systems: (1) H2O; (2) 15% HOAc; (3) BAW
(n-BuOH–HOAc–H2O, 4:1:5, upper layer); (4) C6H6-n-
BuOH–H2O–pyridine (1:5:3:3, upper layer). Solvents 3
and 4 were used for sugar analysis and solvent 3 for
preparative isolation on Whatman no. 3MM paper.
The deshielding of the second resonance is obviously
due to the acylation by p-coumaric acid. Assignment of
the remaining glucose carbons was aided by comparison
with the reported 13C chemical shifts of kaempferol 3,7-
di-O-glucoside (Markham et al., 1978). The presence of
only one p-coumaroyl moiety in 4 followed from the
single carboxyl carbon resonance at ꢀ 166.3 and from
the recognized characteristic pattern of the remaining p-
coumaroyl carbon resonances (see Experimental). The
recorded chemical shifts of the kaempferol carbon reso-
nances, in the 13C NMR spectrum, confirmed substitu-
tion at the 3- and 7-positions. This followed from the
relative upfield shifts of the C-3 and C-7 resonances to ꢀ
133.4 and 162.8, respectively. As expected, these shifts
3.1. Plant material
Flowering specimens of Lotus polyphyllos E. D. Clarke,
shrubs, were collected from the sand dunes west of Mersa

810
A.M. D. El Mousallami et al. / Phytochemistry 60 (2002) 807–811
Metrouh, Egypt, during June 2001and identified by Dr.
M. El Gibali, National Research Centre (NRC), Dokki,
Cairo, Egypt. A voucher specimen has been deposited at
the herbarium of the NRC.
methylenic H-600 proton), 3.3–3.9 (m, glucose protons
overlapped by water protons). 13C NMR of 4: Table 2.
3.4. Quercetin 3-O-ꢁ-(600-E-p-coumaroylglucopyrano-
side)-7-O-b- gucopyranoside (5)
3.2. Isolation and identification
Mr: 772, ESI–MS: negative ion: m/z (rel. int.): 771(60),
[M–H]ꢁ, 463 (28), [M - p-coumaroyl glucose]ꢁ, 301(36),
The ground dried aerial parts (2 kg) of L. polyphyllos
plants were extracted (ꢂ3) by refluxing with 4 l. of
EtOH–H2O (3:1) over a boiling water bath for 8 h. The
conc. extract was applied to a Sephadex LH-20 column
(100ꢂ5 cm inte) and eluted with H2O followed by H2O–
MeOH mixtures of decreasing polarities to yield, nine
fractions, among which two were found to contain fla-
vonoids, eluted by H2O–MeOH (70:30) and (40:60).
They appeared on the column as dark purple bands
under UV light. Repeated PPC chromatography, using
BAW as an eluant afforded pure samples of 1, (105 mg),
2 (164 mg), 3 (88 mg), from the 30% column fraction
and 4 (76 mg), 5 (90 mg) and 6 (58 mg), from the 60%
column fraction.
[quercetin]ꢁ, 145 (33), [p-coumaroyl]ꢁ. Rf-values:
MeOH
max
Table 1. UV l
nm: Table 1. Normal acid hydrolysis
gave glucose (CoPC), quercetin and p-coumaric acid
[CoPC, UV spectral data (Table 1), 1H NMR of quercetin:
ppm 6.22 (d, J=2.5 Hz, H-6), 6.40 (d, J=2.5 Hz, H-8),
6.85 (d, J=7.5 Hz, H-50),. 07.35 (d, J=2.5 Hz, H-20), 7.5
(dd, J=7.5 and 2.5 Hz, H-6 ), 12.3 (s, OH-5)]. 1H NMR of
5: p-coumaroyl moiety: ppm 6.15 (d, J=16 Hz, H-80000),
6.86 (d, J=8 Hz, H-30000 and H-50000), 7.50 (d, J=16 Hz,
H-70000), 7.54 (d, J=8 Hz, H-20000 and H-60000); quercetin
moiety: ppm 6.36 (d, J=2.5, H-6), 6.75 (d, J=2.5 Hz,
H-8), 6.8 (d, J=7.5 Hz, H-50), (m, H-20 and H-60), 12.6
(s, OH-5); b-glucoside moieties: ppm 5.16 (d, J=8.5 Hz,
H-100’), 5.50 (d, J=8.5 Hz, H-100), 4.4 (d, d, J=12 and
4.5 Hz, one methylenic H-600 proton), 4.24 (m, one
methylenic H-600 proton), 3.3–3.9 (m, glucose protons
overlapped by water protons). 13C NMR of 5: Table 2.
3.3. Kaempferol 3-O-ꢁ-(600-E-p-coumaroylglucopyrano-
side)-7-O-ꢁ-glucopyranoside (4)
Mr: 756, ESI–MS: negative ion: m/z (rel. int.): 755(52),
[M–H]ꢁ, 447 (30), [M - p-coumaroyl glucose]ꢁ, 285(39),
3.5. Isorhamnetin 3-O-ꢁ-(600-E-p-coumaroyglucopyrano-
side)-7-O-ꢁ-glucopyranoside (6)
[kaempferol, 145(24),[p-coumaroyl]ꢁ. Rf-values: Table 1.
MeOH
max
UV l
glucose (Co-PC), kaempferol and p-coumaric acid [Co-
nm: Table 1. Normal acid hydrolysis gave
Mr: 786, ESI–MS: negative ion: m/z (rel. int.): 785
(63), [M–H]ꢁ, 477 (40), [M - p-coumaroyl glucose]ꢁ, 31 5
(44), [isorhamnetin aglycone]ꢁ, 145 (32), [p-coumaroyl]ꢁ.
Rf-values: Table 1. UV lMmaexOH nm: Table 1. Normal acid
hydrolysis gave glucose (CoPC), isorhamnetin and p-
coumaric acid [CoPC, UV spectral data (Table 1) and
1H NMR, 1H NMR of isorhamnetin: ppm 6.22 (d, J=2.5
Hz, H-6), 6.46 (d, J=2.5 Hz, H-8), 6.88 (d, J=7.5 Hz, H-
50), 7.7 (d, d, J=7.5 and J=2.5 Hz, H-60), 7.78 (d, J=2.5
1
PC, UV spectral data (Table 1), H NMR of kaemp-
ferol: ppm 6.2 (d, J=2.5 Hz, H-6), 6.42 (d, J=2.5 Hz,
H-8), 6.98 (d, J=7.5 Hz, H-30 and H-50), 8.03 (d, J=7.5
Hz, H-20 and H-60); H NMR of p-coumaric acid: ppm
1
6.3 (d, J=16 Hz, H-80000), 6.8 (d, J=7.5 Hz, H-30 and H-
50), 7.46 (d,=7.5 Hz, H-20 and H-60), 7.55 (d, J=16 Hz,
H-20 and H-60). b-Glucosidase enzymatic hydrolysis
yielded kaempferol 3-O-b-(600-p-coumaroylglucoside):
Rf-values: Table 1. 1H NMR: p-coumaroyl moiety: ppm
6.2 (d, J=16 Hz, H-8), 6.84 (d, J=7.5 Hz, H-30 and H-
50), 7.4 (d, J=16 Hz, H-7), 7.45 (d, J=7.5 Hz, H-20 and
H-60); kaempferol moiety: 6.18 (d, J=2.5 Hz, H-6), 6.38
(d, J=2.5 Hz, H-8), 6.88 (d, J=7.5 Hz, H-30 and H-50),
8.0 (d, J=7.5 Hz, H-20 and H-60), 12.2 (s, OH-5); b-
glucoside moiety: 5.5 (d, J=8.5 Hz, H-1), 4.35 (dd,
J=12 and 3.5 Hz, one proton H-6), 4.18 (m, one pro-
ton H-6), 3.3–3.85 (m, glucose protons overlapped with
water protons). 1H NMR of 4: p-coumaroyl moiety:
ppm 6.15 (d, J=16 Hz, H-80000), 6.8 (d, J=8 Hz, H-30000
and H-50000), 7.55 (d, J=16 Hz, H-70000), 7.6 (d, J=8 Hz,
H-20000 and H-60000); Kaempferol moiety: ppm 6.32 (d,
J=2.5, H-6), 6.72 (d, J=2.5 Hz, H-8), 6.86 (d, J=7.5
Hz, H-30 and H-50), 8.0 (d, J=7.5 Hz, H-20 and H-60),
11.8 (s,OH-5); b-glucoside moieties: ppm 5.02 (d, J=8.5
Hz, H-100’), 5.55 (d, J=8.5 Hz, H-100), 4.26 (d, d, J=12
and 4.5 Hz, one methylenic H-600 proton), 4.12 (m, one
0
1
Hz, H-2 ), 11.8 (s, OH-5)]. H NMR of 5: p-coumaroyl
moiety: ppm 6.24 (d, J=16 Hz, H-80000), 6.88 (d, J=8 Hz,
H-30000 and H-50000), 7.45 (d, J=16 Hz, H-70000), 7.52 (d, J=8
Hz, H-20000 and H-60000); isorhamnetin moiety: ppm 6.33 (d,
J=2.5, H-6), 6.70 (d, J=2.5 Hz, H-8), 6.85 (d, J=7.5 Hz,
H-50), 7.8 (dd, J=2.5 and 7.5 Hz, H-60), 7.8.5 (d, J=2.5
Hz, H-20), 12.3 (s, OH-5); b-glucoside moieties: ppm 5.12
(d, J=8.5 Hz, H-100’), 5.60 (d, =8.5 Hz, H-100), 4.32 (dd,
J=12 and 4.5 Hz, one methylenic H-600 proton), 4.2 (m,
one methylenic H-600 proton), 3.22–3.88 (m, glucose pro-
tons overlapped by water protons). 13C NMR of 5: Table 2.
Acknowledgements
The authors wish to express their gratitude to Dr. J.
Hau, Nestle Research Center Lausanne, CH-1000 Lau-
sanne 26, Switzerland for the ESI-MS measurements.

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811
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