A stilbene and dihydrochalcones with radical scavenging activities from Loiseleuria procumbens

Article abstract of DOI:10.1016/S0031-9422(00)00200-4

A new dihydrochalcone, 6_-Acetylphloridzosid, was isolated from the whole plant of Loiseleuria procumbens (L.) Desv. and identified as 2'-O-(6_-O-acetylglucopyranosyl)-4,4',6'-trihydroxydihydrochalcone by spectroscopic methods. In addition, one stilbene and three other dihydrochalcones were identified as (E)-piceid, phloretin (2',4,4',6'-tetrahydroxydihydrochalcone), phloridzosid (2'-O-glucopyranosyl-4,4',6'-trihydroxydihydrochalcone) and asebotin (2'-O-glucopyranosyl-4'-methoxy-4,6'-dihydroxydihydrochalcone), respectively. Some of these compounds showed scavenging properties towards the 2,2-diphenyl-1-picrylhydrazyl radical and antioxidant properties in a test with lysozyme. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00200-4

Phytochemistry 54 (2000) 871±874
www.elsevier.com/locate/phytochem
A stilbene and dihydrochalcones with radical scavenging activities
from Loiseleuria procumbens
a
Muriel Cuendet , Olivier Potterat , Aline Salvi , Bernard Testa , Kurt Hostettmann *
a
b
b
a,
a
Institut de Pharmacognosie et Phytochimie, Universite de Lausanne, BEP, CH-1015 Lausanne, Switzerland
b
Institut de Chimie TheÂrapeutique, Universite de Lausanne, BEP, CH-1015 Lausanne, Switzerland
Received 25 January 2000; received in revised form 22 May 2000
Abstract
A new dihydrochalcone, 60-acetylphloridzosid, was isolated from the whole plant of Loiseleuria procumbens (L.) Desv. and
identi®ed as 2'-O-(60-O-acetylglucopyranosyl)-4,4',6'-trihydroxydihydrochalcone by spectroscopic methods. In addition, one
stilbene and three other dihydrochalcones were identi®ed as (E)-piceid, phloretin (2',4,4',6'-tetrahydroxydihydrochalcone),
phloridzosid (2'-O-glucopyranosyl-4,4',6'-trihydroxydihydrochalcone) and asebotin (2'-O-glucopyranosyl-4'-methoxy-4,6'-
dihydroxydihydrochalcone), respectively. Some of these compounds showed scavenging properties towards the 2,2-diphenyl-1-
picrylhydrazyl radical and antioxidant properties in a test with lysozyme. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Loiseleuria procumbens; Ericaceae; Radical scavenging activity; Stilbene; Dihydrochalcone; E-piceid; Phloretol; Phloridzosid; Asebotin;
60-Acetylphloridzosid
1. Introduction
phloretic acid, phloridzosid, phloretol and (hydroxy-4-
phenyl)-3-propionic acid (Serve et al., 1985, 1986).
In the course of our search for new antioxidants
from higher plants, particular attention has been given
to plants growing at high altitude (>2000 m). Such
species have to face more intense UV radiation and
may respond to the resulting oxidative stress by produ-
cing a greater diversity of antioxidant molecules. We
detected in the methanolic whole plant extract of Loi-
seleuria procumbens (L.) Desv. (Ericaceae) compounds
which reduced the 2,2-diphenyl-1-picrylhydrazyl
2
. Results and discussion
The methanolic extract of the whole plant was frac-
tionated by a combination of CC on silicagel, medium-
pressure liquid chromatography (MPLC) and gel ®l-
tration on Sephadex LH-20 to provide compounds 1±5
(
see Section 3).
Compound 1 was identi®ed as (E)-piceid (3-O-glu-
(
DPPH) radical in a TLC autographic assay (Cuendet
et al., 1997; Takao et al., 1994). Analysis by HPLC-
UV and TLC (Godin reagent) (Godin, 1954) showed
the presence of dihydrochalcones or ¯avanones.
L. procumbens is a small bush growing in the subal-
pine to alpine regions of Europe and North America.
Previous work on the plant had led to the isolation of
copyranosyl-5,4'-dihydroxystilbene) by comparison of
1
13
its spectroscopic data ( H- and C-NMR, UV, FAB-
MS) with literature values (Wa€o Teguo et al., 1996).
The reaction of compounds 2±5 with Godin reagent
gave an orange color. The H NMR spectra of these
compounds revealed signals for two CH groups, typi-
1
2
cal for a dihydrochalcone. By reference to the litera-
ture (Hu€ord and Oguntimein, 1980), it was possible
to identify compounds 2±4 as phloretin, phloridzosid
and asebotin, respectively.
*
Corresponding author. Tel.: +41-21-6924561; fax: +41-21-
924565.
E-mail address: kurt.hostettmann@ipp.unil.ch (K. Hostettmann).
6
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00200-4

8
72
M. Cuendet et al. / Phytochemistry 54 (2000) 871±874
Compound 5, named 60-acetylphloridzosid, was
also evaluated in a test with lysozyme by a capillary
electrophoretic method (Salvi et al., 1999). Trolox (6-
hydroxy-2,5,7,8-tetraethylchroman-2-carboxylic acid)
and chlorogenic acid were used as reference com-
pounds. Compound 1 presented the highest activity,
but remained less active than trolox and chlorogenic
acid. Compounds 4 and 5 showed modest activity and
2 and 3 had a pro-oxidant e€ect (Table 2).
obtained as a white microcrystalline powder. In its
positive ion D/CI mass spectrum, a peak for the am-
+
monium adduct [M + NH4] was observed at m/z
+
96. The pseudomolecular ion signal [M + H] was
4
at m/z 479. Other fragments at m/z 292 [(M +
+
+
NH )� 204] and 275 [(M + H)� 204] were attribu-
4
ted to the aglycone. The di€erence of 204 amu could
be due to an acetylated hexosyl group (162 amu + 42
amu). The molecular formula C H O was deduced
from the combined D/CI-MS, C and DEPT NMR
data. The dihydrochalcone skeleton was con®rmed by
2
3
26 11
13
3. Experimental
1
the signals for the H-b protons in the H NMR spec-
3.1. General
trum ꢀd 2.88, 2H, t, J ˆ 7:8 Hz) and the C-a and C-b
1
3
carbons in the C NMR spectrum ꢀd 46.83 and
TLC: Silica gel 60 F254 sheets (Merck); CHCl3±
MeOH±H O (65:35:5). Open column chromatography
30.83). All signals obtained for compound 5 were iden-
tical to those of 3, except for the down®eld shift of H-
2
(CC): Sephadex LH-20 (Pharmacia) and silica gel (63±
200 mm, Merck). Medium-pressure liquid chromatog-
raphy (MPLC): home-packed LiChroprep-RP-18 col-
umn (15±25 mm, 460 Â 36 mm i.d., Merck). Anal.
HPLC: Hewlett Packard 1050 instrument equipped
with a photodiode array detector; Nucleosil RP-18 col-
umn (7 mm; 250 Â 4 mm i.d., Macherey-Nagel);
1
13
6
NMR spectra. These data showed an esteri®cation on
0 and the up®eld shift of C-50 in the H and
C
the hydroxyl group at C-60. Two supplementary sig-
13
nals appeared in the C NMR spectrum at d 172.80
and 20.64, corresponding to a carbonyl- and a CH3-
group, respectively; in addition, a singlet at d 2.03 cor-
1
H
responding to an acetyl group appeared in the
MeCN±H O ꢀ5:95450:50 in 30 min, 0.05%
2
NMR spectrum. This signal showed the presence of an
acetyl group at C-60.
Final proof of structure 5 was obtained by alkaline
hydrolysis which a€orded phloridzosid. With the use
of HPLC and TLC co-chromatography, this derivative
was found to be compound 3. Thus, compound 5 is
CF COOH, 1 ml/min). UV: Shimadzu UV-160A and
3
Perkin-Elmer Lambda-3 spectrophotometers. Mp:
Mettler FP-80/82 hot-stage apparatus; uncorrected.
2
D
0
1
13
‰aŠ : Perkin-Elmer 241 polarimeter. H and
C
NMR: Varian Inova 500 spectrometer; d in ppm rel.
to TMS; J in Hz; carbon multiplicities from DEPT ex-
periments. D/CI-MS: Finnigan-MAT TSQ-700 triple-
stage quadrupole instrument. High-performance capil-
lary electrophoresis: Beckman P/ACE Capillary Elec-
trophoresis. Capillary: 50 mm i.d. fused silica capillary,
47 cm in length (40 cm e€ective length up to the detec-
tor). Prior to ®lling a new capillary with running buf-
fer, it was hydrated by high pressure rinse (20 psi), a
10 min rinse with 1 M HCl was followed by a 3 min
6
0-acetylphloridzosid.
Since the methanolic extract of L. procumbens
showed activity in the DPPH TLC assay, the radical
scavenging properties of compounds 1±5 were evalu-
ated against this radical in a spectrometric assay
(
Lamaison et al., 1991). Quercetin and BHT (2,6-
di(tert-butyl)-4-methylphenol) were used as reference
compounds. The activities of 1 and 2 were comparable
to that of BHT, while the other compounds did not
signi®cantly reduce the free radical (Table 1).
The antioxidant activities of compounds 1±5 were
Table 2
Antioxidant activity of compounds 1±5 against lysozyme studied by
capillary electrophoresis
Table 1
Percentage of DPPH radical reduction by compounds 1 and 2 at
a
Compounds
IC50 (mM)
60214:0
Pro-oxidant e€ect
di€erent concentrations. (measurement at 517 nm, determination
after 30 min)
1
2
3
4
5
b
a
Concentration (mM)
Quercetin
BHT
1
2
2225:2
190224:0
100217:0
3128:7
8
4
2
1
0.0
0.0
0.0
0.0
96.6
95.8
84.3
43.8
21.8
11.0
41.3
29.5
14.9
8.1
54.2
33.7
17.9
9.5
33.9
26.0
18.8
12.4
7.6
b
Trolox
Chlorogenic acid
1022:3
a
5
2
.0
.5
3.3
3.3
4.7
2.4
Extrapolated value from measures of 1±50 mM solutions, because
of the pro-oxidant e€ect observed at 100 mM.
Trolox = 6-hydroxy-2,5,7,8-tetraethylchroman-2-carboxylic acid
4.4
b
a
DPPH = 2,2-diphenyl-1-picrylhydrazyl.
BHT = 2,6-di-(tert-butyl)-4-methylphenol.
IC50 represents the concentration inhibiting oxidation by 50%.
Values are expressed as mean2SD for duplicate experiments.
b

M. Cuendet et al. / Phytochemistry 54 (2000) 871±874
873
Table 3
1
a
3
H NMR spectral data of compounds 3 and 5 (measured in CD OD)
Assignment
3
5
H-2
H-3
H-5
H-6
H-a
H-b
H-3'
H-5'
H-10
H-20
H-30
7.06 (1H, d, J ˆ 8:3)
6.69 (1H, d, J ˆ 8:3)
6.69 (1H, d, J ˆ 8:3)
7.06 (1H, d, J ˆ 8:3)
7.07 (1H, d, J ˆ 8:3)
6.70 (1H, d, J ˆ 8:8)
6.70 (1H, d, J ˆ 8:8)
7.07 (1H, d, J ˆ 8:3)
2.87 (2H, t, J ˆ 7:6)
6.18 (1H, d, J ˆ 2:4)
5.97 (1H, d, J ˆ 2:4)
5.05 (1H, d, J ˆ 7:3)
2.88 (2H, t, J ˆ 7:8)
6.15 (1H, d, J ˆ 2:0)
6.00 (1H, d, J ˆ 2:0)
5.05 (1H, d, J ˆ 7:3)
3
.35±3.52 (5H, unresolved, H-20±H-50 and H-a)
3.37±3.50 (4H, unresolved, H-20±H-40 and H-a)
H-40
H-50
Ha-60
Hb-60
Ac-CH
3.65 (1H, ddd, J ˆ 10:0, 6.6, 2.1)
4.40 (1H, dd, J ˆ 12:0, 2.1)
4.27 (1H, dd, J ˆ 12:0, 6.6)
2.03 (3H, s)
3.91 (1H, dd, J ˆ 12:4, 1.9)
3.72 (1H, dd, J ˆ 12:4, 5.4)
3
±
a
Proton coupling constant (J in Hz), signal multiplicities and integrations are in parentheses.
rinse with H O, a 10 min rinse with 1% NaOH, a 3
2
3.4. Reduction of 2,2-diphenyl-1-picrylhydrazyl (2,2-
diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl; DPPH)
radical
min rinse with H O, and ®nally a 10 min rinse with
2
the running bu€er (phosphate bu€er 50 mM pH 3.0).
The running bu€er was ®ltered through a 2 mm ®lter
(
Titan syringe ®lter, Infochroma, Zug, Switzerland).
30 ml of a solution containing the compound to be
tested and 200 ml of MeOH were added to 50 ml of a
0.02% MeOH solution of DPPH. Absorbance at 517
nm was determined after 30 min, and the percent of
activity was calculated.
The temperature of the capillary during electrophoretic
analysis was kept constant at 308C by a liquid thermo-
stating system.
3.2. Plant material
3
.5. Antioxidant activity against lysozyme
Whole plant of L. procumbens (L.) Desv. was col-
lected in Col du Sanetsch (altitude 2250 m), Switzer-
land, in July 1996. A voucher specimen (No. 96162) is
deposited at the Institut de Pharmacognosie et Phyto-
chimie, University of Lausanne, Switzerland.
Lysozyme (1 mg/ml) with antioxidants in various
concentrations were incubated for 20 min, at 4020:18,
in phosphate bu€er (10 mM, pH 7:420:1). The anti-
oxidants were dissolved in MeOH and a small volume
(
5 ml) was added to the protein solution, so as to limit
3
.3. Extraction and isolation
the amount of organic solvent in the assay (total
volume 0.5 ml) to 1% (v/v). The oxidation was in-
itiated by the addition of AAPH (2,2'-azobis(2-amidi-
nopropane)-dihydrochloride) dissolved in phosphate
bu€er.
Protein oxidations were performed in the presence
of 10 mM AAPH with or without antioxidants at
4020:18 during 60 min, with continuous shaking,
under air atmosphere.
Immediately after incubation, the samples were
placed in ice for 10 s in order to suciently decrease
the temperature to stop the free radical generation
from AAPH. 200 ml of the oxidation samples were
mixed with the internal standard to obtain the follow-
ing ®nal concentrations, lysozyme 0.4 mg/ml with 0.3
mg/ml a-chymotrypsinogen A as internal standard.
Samples were placed on the inlet tray of the P/ACE
The dried whole plant (630 g) was ground and
extracted at room temperature successively with
CH Cl ꢀ3 Â 2000 ml) and MeOH ꢀ3 Â 2000 ml) to
2
2
yield 64 and 165 g of extract, respectively. A portion
(70 g) of the MeOH extract was subjected to CC on
silica gel eluted with CHCl ±MeOH (9:1±1:1) to pro-
3
vide 13 fractions (1±13). Fraction 4 was puri®ed by gel
ltration on Sephadex LH-20 CHCl ±MeOH (1:1) to
®
yield phloretin (2; 15 mg). Fractions 7 and 10 were
3
separated by MPLC (RP-18, MeOH±H O 25:75±1:1,
2
¯
p, respectively. Fractions 7c, 10g, 10i and 10n con-
tained pure 5 (950 mg), (E)-piceid (1; 440 mg), phlor-
idzosid (3; 790 mg) and asebotin (4; 21 mg),
respectively.
ow rate 4 ml/min) a€ording fractions 7a±f and 10a±

8
74
M. Cuendet et al. / Phytochemistry 54 (2000) 871±874
Table 4
3
.7. 60-Acetylphloridzosid (5)
13
C NMR spectral data of compounds 3 and 5 (measured in
CD OD)
3
2
D
0
Microcrystalline white powder. ‰aŠ � 378 (MeOH,
c0.5). Mp 103� 1078C. UV lmax (MeOH) nm ꢀlog e):
Carbon no.
3
5
2
23 (4.30), 284 (4.20). D/CI-MS m/z: 496 [M +
+
+
1
133.84
130.31
116.03
156.14
116.03
130.31
46.83
30.73
206.48
106.73
167.18
95.40
165.77
98.33
162.15
101.90
74.62
78.24
70.98
78.36
62.34
±
133.81
130.32
116.05
156.23
116.05
130.32
46.83
NH ] , 479 [M + H] , 292 [(M + NH )±60-O-acetyl-
4
4
2
+
+
1
glucosyl] , 274 [M-60-O-acetylglucosyl] . H- and
C-NMR: Tables 3 and 4.
3
13
4
5
6
a
b
CO
30.83
206.49
106.87
167.31
95.82
Acknowledgements
1
2
3
4
5
6
1
2
3
4
5
6
'
'
'
'
'
Financial support was provided by the Swiss
National Science Foundation.
165.82
98.44
'
162.03
101.92
74.60
0
0
0
0
0
0
78.21
71.31
References
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64.44
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±
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