Phenylvaleric acid and flavonoid glycosides from Polygonum salicifolium

Article abstract of DOI:10.1021/np9900674

(3R)-O-β-D-Glucopyranosyloxy-5-phenylvaleric acid (1), (3R)-O-β-D- glucopyranosyloxy-5-phenylvaleric acid n-butyl ester (2), and a new dihydrochalcone diglycoside 4'-O-[β-D-glucopyranosyl-(1→-6)- glucopyranosyl]-oxy-2'-hydroxy-3',6'-dimethoxydihydrochalco

Full text of DOI:10.1021/np9900674

J . Nat. Prod. 1999, 62, 1101-1105
1101
P h en ylva ler ic Acid a n d F la von oid Glycosid es fr om P olygon u m sa licifoliu m
Ihsan Calis,*,† Ayse Kuruu¨zu¨m,† L. O¨ mu¨r Demirezer,† Otto Sticher,‡ Walter Ganci,§ and Peter Ru¨edi§
Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, TR-061000 Ankara, Turkey,
Swiss Federal Institute of Technology (ETH) Zurich, Department of Pharmacy, CH-8057 Zurich, Switzerland, and
University of Zurich, Institute of Organic Chemistry, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
Received February 22, 1999
(3R)-O-â-D-Glucopyranosyloxy-5-phenylvaleric acid (1), (3R)-O-â-D-glucopyranosyloxy-5-phenylvaleric acid
n-butyl ester (2), and a new dihydrochalcone diglycoside 4′-O-[â-D-glucopyranosyl-(1f6)-glucopyranosyl]-
oxy-2′-hydroxy-3′,6′-dimethoxydihydrochalcone (3), together with six known flavonoid glycosides [kaempferol-
3-O-â-D-glucopyranoside () astragalin) (4), kaempferol-3-O-â-D-galactopyranoside (5), quercetin-3-O-â-
D-glucopyranoside () isoquercitrin) (6), quercetin-3-O-â-D-galactopyranoside () hyperoside) (7), quercetin-
3-O-(2′′-O-galloyl)-â-D-glucopyranoside (8), and quercetin-3-O-â-D-glucuronopyranoside (9)] were isolated
from the aerial parts of Polygonum salicifolium. The structure elucidation of the isolated compounds
was performed by spectroscopic (UV, IR, ESI-MS, 1D- and 2D-NMR), chemical (methylation, enzymatic
hydrolysis, partial synthesis), and chromatographic methods (HPLC, Chiralcel OD). The flavonoid
glycosides (4-9) demonstrated scavenging properties toward the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical in TLC autographic assays.
Ch a r t 1
The genus Polygonum (Polygonaceae) is represented by
thirty-three species in the flora of Turkey.1,2 Some of them
are used in traditional medicine against kidney stones and
as antidiabetic, diuretic, and antidiarrhoeal agents.3 Fla-
vonoids,4 chalcones,5-8 anthraquinones,9 naphthoquinone,9
sesquiterpenoids,10 lignans,11 coumarins,12 stilbene glyco-
side,13 and acetophenone glycosides14 are some of the
secondary metabolites isolated from Polygonum species.
There is only one paper reported on Polygonum salici-
folium, showing the presence of kaempferol-7-O-rhamno-
glucoside, quercetin-7-O-galactoside, orobol-7-O-glucoside,
and isorhamnetin-3-O-galactoside in the leaves, stems, and
flowers of P. salicifolium.15 We now report on the isolation
and structure elucidation of the novel compounds 1-3, in
addition to six known flavonoid glycosides (4-9) from the
aerial parts of P. salicifolium Brouss. ex Willd (Chart 1).
Resu lts a n d Discu ssion
The methanolic extract of the aerial parts of P. salici-
folium was concentrated and suspended in water and
partitioned with solvents of increasing polarity (n-hexane,
diethyl ether, ethyl acetate, and n-butanol). The n-BuOH
residue was fractionated on polyamide and repeated col-
umn chromatography (silica gel, RP-18, Sephadex LH-20)
to yield compounds 1-9.
Compound 1 was obtained as a colorless powder, [R]23
D
-7.5° (c 0.22, MeOH). The molecular formula of 1 was
determined to be C17H24O8 on the basis of negative-ion
ESIMS (m/z 355 [M - H]-, 711 [2M - H]-). In the UV
spectrum of 1, the maximum bands are at 268, 261, 252,
and 248 nm. Its IR spectrum showed absorption bands due
to hydroxy (3369 cm-1), carboxyl (1713 cm-1), aromatic
(1567 cm-1), and ether (1077 and 1033 cm-1) groups.
Sixteen carbons comprising six aromatic carbons, one
hydroxymethine, three methylenes (except for the -COOH
carbon due to an aglycone), and six carbons due to gluco-
pyranose were observed in the 13C NMR spectrum of 1
(Table 1). The 1H NMR and DQF-COSY spectrum of 1
* To whom correspondence should be addressed. Fax: +90 312 3114777.
E-mail: acalis@dominet.in.com.tr.
† Hacettepe University.
revealed the presence of a monosubstituted benzene ring
[δ 7.22 (4H, m, H-2′,3′,5′,6′), 7.12 (1 H, m, H-4′)] and a
‡ Swiss Federal Institute of Technology.
§ University of Zurich.
10.1021/np9900674 CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/14/1999

1102 J ournal of Natural Products, 1999, Vol. 62, No. 8
Calis et al.
Ta ble 1. 13C and 1H NMR Spectral Data for Compounds 1, 1a , 1b, and 2 (13C, 75.5 MHz; 1H, 300 MHz, MeOD) and HMBC
Correlations for 1a
1
1a
1b
2
position C atom δC (ppm) δH (ppm), J (Hz) HMBC (from C to H) δH (ppm), J (Hz) δH (ppm) δC (ppm) δH (ppm), J (Hz)
1
2
C
CH2
181.7
45.2
H-2, H-3
2.41 dd (14.7, 5.5) H-3, H-4
2.64 dd(14.7, 7.0)
173.7
42.7
2.56 dd (15.5, 5.7)
2.80 dd (15.5, 6.8)
4.11 m
1.89 m
2.79 m
2.66 m
2.79 m
4.22 m
1.78 m
2.44 m
2.41 dd (15.3, 6.0)
2.81 dd (15.3, 6.5)
4.08b
3
4
5
CH
CH2
CH2
78.3
37.9
32.4
4.17 m
1.92 m
2.78 m
H-1′′, H-2, H-4
H-2, H-5
H-3, H-4, H-2′, H-6′
78.0
37.9
32.1
1.90 m
2.80b
COOCH3
3.66 s
1′
2′
3′
4′
5′
C
143.0
129.3
129.5
126.7
129.5
129.3
H-5
143.3
129.5
129.3
126.7
129.3
129.5
CH
CH
CH
CH
CH
7.22 m
7.22 m
7.12 m
7.22 m
7.22 m
H-3′, H-4′, H-5
H-4′
H-2′, H-3′, H-5′, H-6′ 7.15 m
H-4′
H-4′
7.23 m
7.23 m
7.20 m
7.20 m
7.20 m
7.20 m
7.20 m
7.23 m
7.23 m
7.17 m
7.23 m
7.23 m
7.23 m
7.23 m
6′
glucose
1′′
2′′
3′′
4′′
CH
CH
CH
CH
CH
CH2
103.8
75.2
78.1
71.6
77.9
62.8
4.41 d (7.7)
3.22b
H-3, H-5′′
H-3′′
H-5′′
4.34 d (7.8)
3.18 dd (7.8, 8.5)
3.32b
104.4
75.2
77.6
71.7
77.4
62.9
4.35 d (7.8)
3.18 dd (7.8, 8.8)
3.36 t (8.8)
3.42b
3.30b
H-3′′
3.27b
3.28b
5′′
6′′
3.28b
3.19b
3.80 dd (11.8, 2.0)
3.60 dd (11.8, 5.0)
3.22b
3.81 dd (12.0, 2.0)
3.63 dd (12.0, 5.5)
3.86 dd (12.0, 2.0)
3.66 dd (12.0, 4.5)
butyl
1′′′
2′′′
3′′′
4′′′
CH2
CH2
CH2
CH3
65.5
20.1
31.7
14.0
4.08 m
1.60 m
1.38 m
0.94 m
a
b
The assignments were based on DEPT, DQF-COSY, HMQC, and HMBC. Signal patterns are unclear due to overlapping.
â-glucopyranosyl moiety [δ 4.41 (d, J )7.7 Hz)]. On the
other hand, three sets of methylene protons [δ 1.92 (2H,
m, H-4], 2.41 (1H, dd, J ) 4.7, 5.5 Hz, H-2a), 2.64 (1 H, dd,
The molecular formula of compound 2 ([R]23 -10.5° (c
D
0.22, MeOH)) was determined to be C21H32O8 on the basis
of positive ion ESIMS (m/z 435 [M + Na]+, 847 [2M +
Na]+). In the UV spectrum of 2, the maximum bands were
observed at 268, 259, 253, and 247 nm. The IR spectrum
showed absorption bands due to hydroxy (3402 cm-1), ester
(1732 cm-1), aromatic (1567 cm-1), and ether groups (1078
J
) 14.7, 7.0 Hz, H-2b), 2.78 (2H, m, H-5)] and one
oxymethine proton at δ 4.17 (1H, m, H-3) were successively
coupled. The 13C NMR signals of 1 were assigned with the
help of an HMQC experiment, establishing direct C-H
bonding. The connectivities of the molecular fragments
were established by a heteronuclear multiple bond correla-
tion experiment (HMBC), where the long-range correlations
were observed between C-1, H-2′; C-1, H-3; C-1′, H-5; C-5,
H-2′; and C-5, H-6′. This experiment also clarified the site
of glycosidation showing a long-range correlation between
the anomeric proton of glucose at δH 4.41 (d, J ) 7.7 Hz,
H-1′′) and the oxygenated carbon atom at δC 78.3 (C-3).
Methylation of 1 with CH2N2 yielded the ester 1a . The
1H NMR spectrum of 1a revealed a methyl resonance (3H)
at δ 3.66 arising from the carbomethoxy group. This result
supported that the presence of a free carboxyl group in 1.
Thus, the constitution of 1 was determined to be 3-O-â-
glucopyranosyloxy-5-phenylvaleric acid. Such a compound
has been isolated recently from Perilla frutescens (Labia-
tae); however, its absolute configuration remained open.16
The absolute configuration at C-3 of 1 was determined
as follows.17 Enzymatic hydrolysis with â-glucosidase
yielded 3-hydroxyphenylvaleric acid (1b) (1H NMR data,
see Table 1). Esterification of 1b with CH2N2 (Et2O) yielded
the hydroxy-ester 1c which was identified unambiguously
as the (R)-enantiomer by HPLC (Chiralcel OD, hexane/2-
propanol 9:1, k′ ) 2.66, ee > 99%). The respective reference
compounds 1c ((+)-(R), k′ ) 2.66), ent-1c ((-)-(S), k′ ) 2.24)
have been prepared by reduction of methyl 3-oxo-5-phen-
ylvaleroate with NaBH4 to yield the racemic mixture (k′ )
2.24 and 2.66) and enantioselective hydrogenation18 with
(R)-BINAP-Ru/H2 afforded 1c and (S)-BINAP-Ru/H2
yielded ent-1c.17 Moreover, the substrate specificity of
â-glucosidase confirms the expected D-series of the glucose
moiety. Consequently, the structure of 1 was established
as (3R)-O-â-D-glucopyranosyloxy-5-phenylvaleric acid.
1
and 1033 cm-1). The H and 13C NMR spectra of 2 (Table
1) were similar to those of 1 except for the presence of
signals due to a butyl moiety. A COSY experiment showed
the proton resonances in the same spin system which were
assigned to a n-butyl moiety: δ 0.94 (t, 3H J ) 7.3 Hz,
Me-4′′′); 1.38 (m, 2H, H2-3′′′); 1.60 (m, 2H, H2-2′′′); 4.08 (t,
2H, J ) 6.6 Hz, H2-1′′′). The HMQC experiment correlated
all proton resonances with those of the corresponding
carbons of the butyl moiety (δ 65.5 t, 20.1 t, 31.7 t, and
14.0 q; C-1′′′-C-4′′′, respectively). The information concern-
ing the linkage of the n-butyl moiety was obtained from
the HMBC spectrum. The long-range correlations were
observed from the following pairs: C-1 (δ 173.7)/H2-1′′′ (δ
4.08) and C-1/H2-2 (δ 2.81 and 2.55). Furthermore, the long-
range correlations between C-3 (δ 78.0) and H-1′′ (δ 4.35),
and C-1′ (δ 143.3) and H2-5 (δ 2.80) supported the proposed
assignments. Therefore, the structure of 2 was established
to be (3R)-O-â-D-glucopyranosyloxy-5-phenylvaleric acid
butyl ester. Since we used n-BuOH during the extraction
procedure, compound 2 is considered to be an artifact.
Compound 3 was obtained as an amorphous, pale yellow
powder, [R]23 -20.0° (c 0.12, MeOH). The elemental
D
formula of 3 was determined as C29H38O5 by negative and
positive ion ESIMS (m/z 625 [M - H]-, and 649 [M + Na]+,
respectively), 1H and 13C NMR spectral data. The complete
1H and 13C connectivity was established by extensive use
and interpretation of 2D (1H-1H) COSY, HMQC (one-bond
13C-1H correlation), and HMBC (long range 13C-1H cor-
relation) NMR spectra. This provided unequivocally the
atomic network for 3 (Table 2). The UV spectrum of 3
showed absorption bands at 333, 287, 233 (sh), and 208
nm. The IR spectrum of 3 showed absorbances for hydroxy

Phenylvaleric Acid and Flavonoid Glycosides from P. salicifolium
J ournal of Natural Products, 1999, Vol. 62, No. 8 1103
Ta ble 2. 13C and 1H NMR Spectral Data for Compound 3 (13C, 75.5 MHz; 1H, 300 MHz, MeOD)a
position
C atom
δC (ppm)
δH (ppm), J (Hz)
HMBC (form C to H)
1
2
3
4
5
6
R
â
C
142.9
129.4
129.4
127.0
129.4
129.4
47.4
H-2, H-6, H2-R, H2-â
H2-â
CH
CH
CH
CH
CH
CH2
CH2
C
C
C
C
C
7.20 m
7.20 m
7.14 m
7.20 m
7.20 m
3.31b
H-3, H-5
H2-â
31.7
2.96 t (7.8)
CdO
207.0
108.3
162.8
132.3
157.3
91.3
159.9
61.5
56.8
H2-R, H2-â
H-5′
1′
2′
3′
4′
5′
6′
H-5′, OMe (C-3′)
H-1′′, Η-5′
CH
C
CH3
CH3
6.37 s
OMe (C-6′), Η-5′
OMe (C-3′)
OMe (C-6′)
3.79 s
3.90 s
glucose
1′′
2′′
3′′
4′′
CH
CH
CH
CH
CH
CH2
101.1
74.7
78.1
71.3
77.6
70.3
5.07 d (7.2)
3.52b
3.30b
3.38b
5′′
6′′
3.77b
4.16 br d (11.8)
H-1′′′
H2-6′′
3.80b
terminal glucose
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
CH
CH
CH
CH
CH
CH2
105.1
75.1
78.0
71.5
77.8
62.7
4.31 d (7.7)
3.16 dd (7.7, 8.5)
3.22b
3.24b
3.50b
3.82 dd (11.8, 2.0)
3.63 dd (11.8, 5.2)
a
b
The assignments were based on DEPT, DQF-COSY, HMQC, and HMBC. Signal patterns are unclear due to overlapping.
(3436 and 3400 cm-1), a conjugated ketone (1625 cm-1),
aromatic (1457 and 1420 cm-1), and ether groups (1068 and
1
1053 cm-1). The H NMR spectrum displayed the charac-
teristic ABX pattern of a monosubstituted benzyl moiety,
five proton multiplets (δ 7.20-7.14, m, H-2,3,4,5,6) char-
acteristic of an unsubstituted B ring, two methoxy reso-
nances at δ 3.90 and 3.79, an aromatic proton at δ 6.37
(1H, s, H-5′), and two methylene signals linked to each
other at δ 3.31 (H2-R) and 2.96 (H2-â) (each 2H, the former
overlapped, the latter t, J ) 7.8 Hz). Additionally, two
anomeric proton resonances were observed at δ 5.07 (d, J
) 7.2 Hz) and 4.31 (d, J ) 7.7 Hz), indicating its diglyco-
sidic dihydrochalcone structure. The 13C NMR spectrum
of 3 exhibited 29 carbon resonances: 7 quaternary carbons
(C), 16 methine (CH), 4 methylene (CH2), and 2 methyl
(CH3). The 1H and 13C NMR spectral data together with
ESIMS supported the presence of two hexose units in 3.
From the chemical shift values and the coupling constants
of the anomeric protons, their glycosidations were found
to be on a phenolic and an aliphatic hydroxyl group,
respectively, and â-linkages for both. The remaining carbon
resonances for the aglycone moiety supported that the
aglycone moiety of 3 has a pentasubstituted A ring as in
dihydropashanone19 except for the different substitution
pattern, two methoxy and two hydroxy groups of which one
of the latters was glycosylated with a disaccharide. The
placement of all substituents on the A ring was established
using 2D 13C-1H long-range correlation (HMBC) and
ROESY NMR experiments. The long-range correlation
between the quaternary carbon atom at δ 157.3 (C-4′) and
the anomeric proton (δ 5.07) of the inner glucose moiety
showed the site of glycosidation. In addition, to the carbon
resonance assigned as C-4′ (δ 157.3), the resonances at δ
108.3 (C-1′), 132.3 (C-3′), 159.9 (C-6′) showed also the long-
range correlations to the aromatic proton at δ 6.37 (H-5′).
F igu r e 1. Heteronuclear multiple-bond correlations for 3. Arrows
point from carbon to proton.
The carbon resonances at δ 132.3 (C-3′) and 159.9 (C-6′)
exhibited long-range correlations to the methoxy signals
at δ 3.79 and 3.90, respectively, showing the site of these
groups on the A ring. The other significant correlations are
shown on Figure 1. Additionally, in the ROESY experi-
ment, the anomeric proton (H-1′′) of the inner glucose and
the methoxyl signal (δH 3.90) located at C-6′ exhibited
correlations to the aromatic proton at δ 6.37 (H-5′), sup-
porting the proposed substitution in the A ring. Apart from
the anomeric protons, 2D NMR experiments (COSY and
HMQC) indicated the presence of two glucose units. A
HMBC experiment performed with 3, showed a correlation
between the anomeric proton at δ 4.31 (H-1′′′) and the
carbon resonans at δ 70.3 (CH2, H-6′′), indicating the
presence of a 6-O-â-D-glucopyranosyl-glucose moiety as the
disaccharidic sugar chain. Reversed correlations between
the anomeric carbon of the terminal glucose moiety at δ
105.1 (C-1′′′) and the methylene protons of the inner glucose
moiety at δ 4.16 and 3.80 (H2-6′′) supported this proposal.
The negative and positive ion ESI mass spectra of 3
exhibited ions at m/z 301 [aglycone (C17H18O5) - H]- and
303 [aglycone (C17H18O5) + H]+ were in good agreement
for the proposed structure of the dihydrochalcone moiety.
Consequently, the structure of compound 3 was established
as 4′-O-[â-D-glucopyranosyl-(1f6)-glucopyranosyl]oxy-2′-

1104 J ournal of Natural Products, 1999, Vol. 62, No. 8
Calis et al.
volume 100 mL) to yield 22 fractions which were combined
into seven main fractions (A-G).
hydroxy-3′,6′-dimethoxydihydrochalcone for which salici-
folioside A is proposed as the trivial name.
The fraction eluted with 10% MeOH (fraction A, 2.1 g) was
chromatographed over CC using normal-phase silica gel (200
g) as stationary phase eluting with CHCl3/MeOH mixtures,
80:20 (200 mL), 70:30 (200 mL), 60:40 (200 mL), 50:50 (200
mL), 40:60 (200 mL), 30:70 (200 mL), and 10:90 (500 mL) to
give 17 fractions (100 mL/fraction) which were combined to
seven main groups on the basis of their TLC profiles. The
fractions eluted with 60% MeOH in CHCl3 was rechromato-
graphed using MPLC (column dimensions 18.5 × 352 mm,
LiChroprep RP-18) eluting with increasing amounts of MeOH
in H2O (H2O, 200 mL; 10% MeOH, 200 mL; 20% MeOH, 200
mL; 30% MeOH, 200 mL; 40% MeOH, 200 mL; MeOH, 200
mL) to give 80 fractions (15 mL/fraction). Fractions 31-41 gave
compound 1 (177 mg). The fractions eluted with 20-30%
MeOH (fraction B, 320 mg) were purified by repeated open
CC (normal-phase silica gel) using CHCl3/MeOH (90:10)
solvent system to yield compound 2 (31 mg). The fraction
eluted with 40-50% MeOH (fraction C, 178 mg) was chro-
matographed on a normal-phase silica gel column (20 g)
eluting with EtOAc (500 mL); EtOAc/MeOH (100:5; 500 mL);
EtOAc/MeOH/H2O (100:5:1; 400 mL); and MeOH (100 mL),
respectively (10-15 mL/fraction). The combined fractions 51-
91 (54 mg) were further fractionated by normal phase silica
gel (10 g) CC using CHCl3/MeOH (90:10; 400 mL; 8 mL/
fraction) to give compound 3 (fractions 26-52; 17 mg).
Fraction E eluted with 70% MeOH (607 mg) was repeatedly
chromatographed over Sephadex LH-20 open CC using MeOH
and normal-phase silica gel open CC using CHCl3/MeOH/H2O
mixtures (90:10:1, 80:20:2, 70:30:3) to yield four flavonoid
glycosides (4-7). Chromatography of fraction G eluted with
90% MeOH (604 mg) using similar conditions yielded com-
pounds 8 and 9.
The presence of dihydrochalcones as well as their gly-
cosides in nature are very rare. Although there are some
studies reporting methoxylated â-hydroxychalcone deriva-
tives from Polygonum nepalense,5 dihydrochalcone and
chalcone derivatives from P. lapathifolium,6,7 there is only
one report on dihydrochalcone monoglycosides from Polygon-
um species, Polygonum senegalense.8
The structures of the six known flavonoids, kaempferol-
3-O-â-D-glucopyranoside () astragalin) (4),20 kaempferol-
3-O-â-D-galactopyranoside (5),21 quercetin-3-O-â-D-glucopy-
ranoside ()isoquercitrin) (6),20 quercetin-3-O-â-D-galac-
topyranoside () hyperoside) (7),20 quercetin-3-O-(2′′-O-
galloyl)-â-D-glucopyranoside (8),22 and quercetin-3-O-â-D-
glucuronopyranoside (9),23 were identified on the basis of
comparison of their spectroscopic (NMR, FABMS) data in
comparison with literature values.
Radical-scavenging properties of the compounds (1-9)
were evaluated against the DPPH radical.24,25 By using
DPPH as a TLC spray reagent, compounds 4-9 (2, 4, 6, 8
µg) appeared as yellow spots against a purple background,
while compounds 1-3 did not react with the radical.
Compounds 6-9, the quercetin glycosides, were more active
in all concentrations applied, while the kaempferol glyco-
sides 4 and 5 showed lower activity. These results indicate
that ortho-hydroxyl groups are an essential feature for the
antioxidant properties of the flavonoid type compounds.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. UV spectra were
determined in spectroscopic grade MeOH on a Shimadzu UV-
160A spectrophotometer. IR spectra were determined on a
Perkin-Elmer 2000 FT-IR spectrometer as pressed KBr disks.
NMR spectra were recorded using a Bruker AMX300 instru-
ment at 300 MHz for 1H and 75.5 MHz for 13C. Complete
proton and carbon assignments are based on 1D (1H, 13C, and
DEPT) and 2D (1H-1H COSY, 1H-13C HMQC, and 1H-13C
HMBC) NMR experiments. ESI-MS was recorded on Hitachi-
Perkin-Elmer-RMUGM mass spectrometer. TLC was carried
out on precoated silica gel 60F-254 aluminum sheets (Merck).
For column chromatography (CC), normal phase silica gel 60
(0.063-0.200 mm, Merck), reversed phase silica gel (LiChro-
prep RP-18, Merck), Sephadex LH-20 (Fluka), and polyamide
(Polyamid-MN-Polyamid SC 6, Macherey-Nagel, Du¨ren) were
used. Compounds were detected by UV fluorescence and/or
spraying with vanillin-H2SO4 reagent followed by heating at
100 °C for 5-10 min and/or exposure to NH3 vapor. For
enzymatic hydrolysis, â-glucosidase from almonds (Emulsin,
Fluka, Nr. 49289) was used. HPLC analyses of the (3R)- and
(3S)-isomers of 3-hydroxyphenylvaleric acid were performed
on Chiralcel OD (250 × 4.6 mm) using hexane/2-propanol (9:
1) as eluent, flow rate 1 mL/min. For the radical-scavenging
TLC autographic assays, 2,2-diphenyl-1-picrylhydrazyl (DPPH,
Fluka) was used as spray reagent.
P la n t Ma ter ia l. P. salicifolium Brouss. ex Willd. was
collected from Trabzon-Uzungo¨l (North Anatolia) in J uly, 1994.
A voucher specimen has been deposited in the Herbarium of
Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe
University (HUEF 94-105).
Extr a ction a n d Isola tion . The dried powdered aerial parts
of P. salicifolium (300 g) were extracted twice with MeOH (2
× 3.5 L) at 40 °C. The MeOH extracts were combined and
evaporated to dryness in vacuo. The crude extract (42 g) was
suspended in water and partitioned with n-hexane, diethyl
ether, ethyl acetate, and n-butanol, respectively. The n-BuOH
extract (7 g) was chromatographed over polyamide (100 g),
eluting with H2O (200 mL), followed by increasing concentra-
tions of MeOH in H2O (10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, and 100% MeOH; each mixture 200 mL; fraction
(3R)-O-â-D-Glu copyr an osyloxy-5-ph en ylvaler ic acid (1):
colorless powder; [R]23D -7.5° (c 0.22, MeOH); UV (MeOH) λmax
248, 252, 261, 268 nm; IR (KBr) νmax 1033, 1077, 1567, 1713,
3369 cm-1; 1H NMR (CD3OD, 300 MHz) see Table 1; 13C NMR
(CD3OD, 75.5 MHz) see Table 1; ESIMS m/z 355 [M - H]-,
711 [2M - H]-.
Meth yla tion of 1. Compound 1 (10 mg) was treated with
CH2N2/Et2O to yield the ester 1a (6 mg) which was purified
on silica gel (10 g) using CHCl3-MeOH-H2O (80:20:2) as
1
eluent (3 mL/fraction). H NMR data, see Table 1.
En zym a tic Hyd r olysis a n d Deter m in a tion of th e Ab-
solu te Con figu r a tion of 1. A solution of 1 (9 mg) in acetate
buffer (pH 4.4, 10 mL) was treated with â-glucosidase (20 mg),
and the solution was left at 37 °C for 48 h. The reaction
solution was evaporated to dryness, and the residue was
chromatographed on silica gel (10 g), using CH2Cl2/MeOH/H2O
1
(90:10:1) to afford 1b (4 mg). H NMR data, see Table 1.
The hydroxy-acid 1b was methylated with CH2N2/Et2O and
purified on silica gel (hexane/AcOEt 2:1) to yield 1c (3 mg).
HPLC on Chiralcel OD, with hexane/2-propanol (9:1) exhibited
only one peak (k′ ) 2.66, ee >99%).
Enantioselective hydrogenation of methyl 3-oxo-5-phenyl-
valeroate (188 mg) with (R)-BINAP-Ru/H2 (30 bar, 100 °C,
24 h) in ethanol yielded after usual workup and chromatog-
raphy on silica gel (hexane/Et2O 2:1) 1c (170 mg), [R]23
)
D
+1.5° (c 2.9, CH2Cl2). HPLC on Chiralcel OD as above eluted
the major enantiomer at k′ ) 2.66 and the minor ent-1c at k′
) 2.24 (ee ) 72%). For a full account on these transformations
that unambiguously established the absolute configuration, see
ref 17.
(3R)-O-â-D-Glu cop yr a n osyloxy-5-p h en ylva ler ic a cid n -
bu tyl ester (2): [R]23 -10.5° (c 0.22, MeOH); UV (MeOH)
D
λmax 247, 253, 259, 268 nm; IR (KBr) νmax 1033, 1078, 1567,
1713, 3402 cm-1; 1H NMR (CD3OD, 300 MHz) see Table 1; 13
C
NMR (CD3OD, 75.5 MHz) see Table 1; ESIMS m/z 435 [M +
Na]+, 847 [2M + Na]+.
Sa licifoliosid e A (3): amorphous, pale yellow powder;
[R]23 -20.0° (c 0.12, MeOH); UV (MeOH) λmax 208, 233 (sh),
D
287, 333 nm; IR (KBr) νmax 1053, 1068, 1420, 1457, 1625, 3400,
3436 cm-1; 1H NMR (CD3OD, 300 MHz) and 13C NMR (CD3OD,
75 MHz), see Table 2; Negative ion ESIMS m/z 625 [M - H]-,

Phenylvaleric Acid and Flavonoid Glycosides from P. salicifolium
J ournal of Natural Products, 1999, Vol. 62, No. 8 1105
301 [aglycone (C17H18O5) - H]-; Positive ion ESIMS m/z 649
[M + Na]+, 303 [aglycone (C17H18O5) + H]+.
Ka em p fer ol-3-O-â-D-glu cop yr a n osid e () a st r a ga lin )
(4): 1H NMR (300 MHz) and 13C NMR (75.5 MHz) data
superimposable with those reported in the literature.20
Ka em p fer ol-3-O-â-D-ga la ctop yr a n osid e (5): 1H NMR
(300 MHz) and 13C NMR (75.5 MHz) data superimposable with
those reported in the literature.21
Qu er cetin -3-O-â-D-glu cop yr a n osid e () isoqu er citr in )
(6): 1H NMR (300 MHz) and 13C NMR (75.5 MHz) data
superimposable with those reported in the literature.20
Qu er cetin -3-O-â-D-ga la ctop yr a n osid e () h yp er osid e)
(7): 1H NMR (300 MHz) and 13C NMR (75.5 MHz) data
superimposable with those reported in the literature.20
Qu er cetin -3-O-(2′′-O-ga lloyl)-â-D-glu cop yr a n osid e (8):
1H NMR (300 MHz) and 13C NMR (75.5 MHz) data super-
imposable with those reported in the literature.22
(6) Ahmed, M.; Khaleduzzaman, M.; Rashid, M. A. Phytochemistry 1988,
27, 2359-2360.
(7) Ahmed, M.; Khaleduzzaman, M.; Islam, M. S. Phytochemistry 1990,
20, 2009-2011.
(8) Maradufu, A.; Ouma, J . H. Phytochemistry 1978, 17, 823-824.
(9) Kimura, Y.; Kozawa, M.; Baba, K.; Hata, K. Planta Medica 1983, 48,
164-168.
(10) Fukuyama, Y.; Sato, T.; Miura, I.; Asakawa, Y. Phytochemistry 1985,
24, 1521-1524.
(11) Kim, H. J .; Woo, E.-R.; Park, H. J . Nat. Prod. 1994, 57, 581-586.
(12) Petrescu, A. D. Plant. Med. Phytother. 1974, 8, 224-227.
(13) Layatilake, G. S.; J ayastiriya, H.; Lee, E. S.; Koonchanok, N. M.;
Geahlen, R. L.; Ashendel, C. L.; McLaughlin, J . L.; Chang, C. J . J .
Nat. Prod. 1993, 56, 1805-1810.
(14) Yoshizaki, M.; Fujino, H.; Arise, A.; Ohmiira, K.; Arisawa, M.; Morita,
N. Planta Medica 1987, 52, 273-275.
(15) Balbaa, S. I.; Afifi, M. S. Bull. Pharm. Sci., Assiut Univ. 1988, 11,
176-184.
(16) Fujita, T.; Terato, K.; Nakayama, M. Biosci. Biotech. Biochem 1996,
60, 732-735.
(17) Calis, I.; Kuruu¨zu¨m, A.; Ganci, W.; Ru¨edi, P. Tetrahedron Asymmetry
1999, 10 (submitted).
(18) Kitamura, M.; Ohkuma, T.; Inoue, S.; Sayo, N.; Kumobayashi, H.;
Akutagawa, S.; Ohta, T.; Takaya, H.; Noyori, R. J . Am. Chem. Soc.
1988, 110, 629-631 and references therein.
(19) Ichino, K.; Tanaka, H.; Ito, K.; Tanaka, T.; Mizuno, M. J . Nat. Prod.
1988, 51, 915-917.
(20) Markham, K. R.; Geiger, H. In The flavonoids: Advances in research
since 1986; Harborne, J . B., Ed.; Chapman & Hall: London, 1993;
Chapter 10, pp 441-447.
Qu er cetin -3-O-â-D-glu cu r on op yr a n osid e (9): 1H NMR
(300 MHz) and 13C NMR (75.5 MHz) data superimposable with
those reported in the literature.23
Rea ction of DP P H Ra d ica l. TLC Au togr a p h ic Assa y.
After developing and drying, TLC plates were sprayed with a
0.2% DPPH solution in MeOH. The plates were examined 30
min after spraying. Active compounds appear as yellow spots
against a purple background.24,25 Quercetin was used as
reference compound.
(21) Agrawal, P. K.; Rastogi, R. P. Heterocycles 1981, 16, 2181-2236.
(22) Kuroyanagi, M.; Yamamoto, Y.; Fukushima, S.; Ueno, A.; Noro, T.;
Miyase, T. Chem. Pharm. Bull. 1982, 30, 1602-1608.
(23) Nawwar, M. A. M.; Souleman, A. M. A.; Buddrus, J .; Linscheid, M.
Phytochemistry 1984, 23, 2347-2349.
(24) Takao, T.; Kitatani, F.; Watanabe, N.; Yagi, A.; Sakata, K. Biosci.
Biotechnol. Biochem. 1994, 58, 1780-1783.
Refer en ces a n d Notes
(1) Davis, P. H. Flora of Turkey and the East Aegean Islands; University
Press: Edinburgh, 1967; Vol. 2.
(2) Davis, P. H. Flora of Turkey and the East Aegean Islands; University
Press: Edinburgh, 1988; Vol 10.
(3) Baytop, T. Therapy with Medicinal Plants (Past and Present); Istanbul
University Publications: Istanbul, 1984; p 313.
(4) Isobe, T.; Noda, Y. Yakugaku Zasshi 1987, 107, 1001-1004.
(5) Rathore, A.; Sharma, S. C.; Tandon, J . S. J . Nat. Prod. 1987, 50, 357-
359.
(25) Cuendet, M.; Hostettmann, K.; Potterat, O.; Dyatmiko, W. Helv. Chim.
Acta 1997, 80, 1144-1152.
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